Plant epsp synthases and methods of use

ABSTRACT

Compositions and methods comprising polynucleotides and polypeptides having EPSP (5-enolpyruvylshikimate-3-phosphate) synthase (EPSPS) activity are provided. In specific embodiments, the sequence has an improved property, such as, but not limited to, improved catalytic capacity in the presence of the inhibitor, glyphosate. Further provided are nucleic acid constructs, plants, plant cells, explants, seeds and grain having the EPSPS sequences. Various methods of employing the EPSPS sequences are provided. Such methods include methods for producing a glyphosate tolerant plant, plant cell, explant or seed and methods of controlling weeds in a field containing a crop employing the plants and/or seeds disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/764,388, filed Mar. 29, 2018, now U.S. Pat. No. 10,655,141, which isa National Phase application of PCT/US16/54399, filed Sep. 29, 2016,which claims the benefit of U.S. Provisional Application No. 62/234,818,filed Sep. 30, 2015, the entire contents of which are herebyincorporated by reference.

FIELD

The field relates to the field of molecular biology. More specifically,it pertains to sequences that confer tolerance to glyphosate.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file namedBB2501PCT_SequenceListing_ST25.txt created on Sep. 19, 2016 and having asize 96 kilobytes and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND

EPSP (5-enolpyruvylshikimate-3-phosphate) synthase is an enzyme thatcatalyzes the conversion of phosphoenolpyruvate and 3-phosphoshikimateto phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP), and itparticipates in the biosynthesis of the aromatic amino acidsphenylalanine, tyrosine, and tryptophan. Glyphosate, the top sellingherbicide in the world, acts a competitive inhibitor forphosphoenolpyruvate.

Glyphosate tolerant crops have been created by introducingglyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS) enzymes into plants. In one example, maize event NK603 usesEPSPS from Agrobacterium sp. strain CP4. The enzyme is highlyinsensitive to inhibition by glyphosate while retaining catalyticefficiency similar to native plant enzymes (Sikorski and Gruys. 1997.Acc. Chem. Res. 30:2-8). In another example, maize event GA21 uses adouble mutant maize EPSPS in which threonine at position 103 is changedto isoleucine and proline at position 107 is changed to serine.

Plant EPSP synthases having kinetic properties that provide adequatetolerance to glyphosate and catalytic capacity to sustain normal ratesof metabolic flux are desired.

SUMMARY

Plant EPSP synthases (herein referred to as EPSPS) and thepolynucleotides that encode them are provided herein. Methods forgenerating glyphosate tolerant plants that are tolerant to the plantEPSPS enzymes are also provided.

Polynucleotides are provided herein that encode plant EPSPS polypeptidesthat comprise G102A and at least one or more amino acid mutationsselected from the group consisting of: (a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS polypeptide comprises a sequencethat is at least 90% identical to SEQ ID NO:2. In some embodiments, thepolynucleotides encode plant EPSPS polypeptides that comprise G102A andat least two or more, three or more, or four or more amino acidmutations selected from the group consisting of (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS polypeptide comprises a sequencethat is at least 90% identical to SEQ ID NO:2.

In other embodiments, the polynucleotide encodes a plant EPSPSpolypeptide that comprises A4W, H54M, L98C, G102A, K173R, I208L, K243E,E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q, and F442V. Instill other embodiments, the polynucleotide encodes a plant EPSPSpolypeptide that comprises A2R, A4W, A72Q, K84R, L98C, G102A, I208L,T279A, E302S, T361S, E391G, D402G, A416G, V438R, and T441Q. In stillother embodiments, the polynucleotide encodes a plant EPSPS polypeptidethat comprises A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, andD402G. In still other embodiments, the polynucleotide encodes the plantEPSPS polypeptide set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

Also provided are recombinant DNA constructs comprising thepolynucleotides disclosed herein; plant cells comprising in theirgenomes a polynucleotide disclosed herein or a recombinant DNA constructcomprising such; and plants comprising in their genomes a polynucleotidedisclosed herein or a recombinant DNA construct comprising such. In someembodiments, the plant cell is a maize cell. In some embodiments, theplant is maize.

Methods of generating glyphosate tolerant plants are provided herein.The methods comprise expressing in a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes aplant EPSPS polypeptide that comprises G102A and at least one amino acidmutation selected from the group consisting of: (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS polypeptide comprises a sequencethat is at least 90% identical to SEQ ID NO:2; and generating aglyphosate tolerant plant that comprises in its genome the recombinantDNA construct. In some embodiments, the methods include expressing in aplant cell a recombinant DNA construct comprising a polynucleotideencoding a plant EPSPS polypeptide comprising G102A and at least two, atleast three, or at least four amino acid mutations selected from thegroup consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f)L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S,(m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s)T441Q, and (t) F442V, wherein each amino acid position corresponds tothe amino acid position set forth in SEQ ID NO:1 and wherein the plantEPSPS polypeptide comprises a sequence that is at least 90% identical toSEQ ID NO:2.

In other embodiments, the method comprises expressing in a plant cell arecombinant DNA comprising a polynucleotide that encodes a plant EPSPSpolypeptide that comprises A4W, H54M, L98C, G102A, K173R, I208L, K243E,E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q, and F442V. Instill other embodiments, the method comprises expressing in a plant cella recombinant DNA comprising a polynucleotide that encodes a plant EPSPSpolypeptide that comprises A2R, A4W, A72Q, K84R, L98C, G102A, I208L,T279A, E302S, T361S, E391G, D402G, A416G, V438R, and T441Q. In stillother embodiments, the method comprises expressing in a plant cell arecombinant DNA comprising a polynucleotide that encodes a plant EPSPSpolypeptide that comprises A2R, A4W, K84R, L98C, G102A, I208L, K243E,E391P, and D402G. In still other embodiments, the method comprisesexpressing in a plant cell a recombinant DNA comprising a polynucleotidethat encodes the plant EPSPS polypeptide set forth in SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6.

Methods of generating glyphosate tolerant plants are provided herein, inwhich an endogenous plant EPSPS gene (in a plant cell) is modified toencode a glyphosate tolerant EPSPS protein that comprises G102A and atleast one amino acid mutation selected from the group consisting of: (a)A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h)I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G,(o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid mutation position corresponds to the amino acidposition set forth in SEQ ID NO:1 and wherein the endogenous plant EPSPSgene encodes a polypeptide comprising a sequence that is at least 90%identical to SEQ ID NO:2; and a glyphosate tolerant plant is grown fromthe plant cell. In some embodiments the modified endogenous plant EPSPSgene encodes a glyphosate tolerant EPSPS protein that comprises G102Aand at least two, at least three, or at least four of the amino acidmutations selected from the group consisting of: (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the endogenous plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:2.

In other embodiments, the modified endogenous plant EPSPS gene encodes aglyphosate tolerant EPSPS protein that comprises: A4W, H54M, L98C,G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R,S440R, T441Q, and F442V. In still other embodiments, the modifiedendogenous plant EPSPS gene encodes a glyphosate tolerant EPSPS proteinthat comprises: A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A, E302S,T361S, E391G, D402G, A416G, V438R, and T441Q. In still otherembodiments, the modified endogenous plant EPSPS gene encodes aglyphosate tolerant EPSPS protein that comprises A2R, A4W, K84R, L98C,G102A, I208L, K243E, E391P, and D402G. In still other embodiments, themodified endogenous plant EPSPS gene encodes a glyphosate tolerant EPSPSprotein that comprises the plant EPSPS polypeptide set forth in SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6.

The endogenous plant EPSPS gene may be modified by a CRISPR/Cas guideRNA-mediated system, a Zn-finger nuclease-mediated system, ameganuclease-mediated system, or an oligonucleobase-mediated system.

Polynucleotide constructs that provide a guide RNA in a plant cell areprovided herein in which the guide RNA targets an endogenous EPSPS geneof the plant cell and the polynucleotide construct further comprises oneor more polynucleotide modification templates to generate a modifiedendogenous EPSPS gene that encodes a plant EPSPS polypeptide comprisingG102A and at least one amino acid mutation selected from the groupconsisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C,(g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m)E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q,and (t) F442V, wherein each amino acid mutation position corresponds tothe amino acid position set forth in SEQ ID NO:1 and wherein theendogenous plant EPSPS gene encodes a polypeptide comprising a sequencethat is at least 90% identical to SEQ ID NO:2. In some embodiments, thepolynucleotide construct comprises one or more polynucleotidemodification templates to generate a modified endogenous EPSPS geneencoding a plant EPSPS polypeptide that comprises G102A and at leasttwo, at least three, or at least four amino acid mutations selected fromthe group consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R,(f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l)T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R,(5) T441Q, and (t) F442V, wherein each amino acid position correspondsto the amino acid mutation position set forth in SEQ ID NO:1 and whereinthe endogenous plant EPSPS gene encodes a polypeptide comprising asequence that is at least 90% identical to SEQ ID NO:2.

In other embodiments, the polynucleotide construct comprises one or morepolynucleotide modification templates to generate a modified endogenousEPSPS gene encoding a plant EPSPS polypeptide that comprises: A4W, H54M,L98C, G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G,V438R, S440R, T441Q, and F442V. In still other embodiments, thepolynucleotide construct comprises one or more polynucleotidemodification templates to generate a modified endogenous EPSPS geneencoding a plant EPSPS polypeptide that comprises: A2R, A4W, A72Q, K84R,L98C, G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G, V438R, andT441Q. In still other embodiments, the polynucleotide constructcomprises one or more polynucleotide modification templates to generatea modified endogenous EPSPS gene encoding a plant EPSPS polypeptide thatcomprises: A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, and D402G.In still other embodiments, the polynucleotide construct comprises oneor more polynucleotide modification templates to generate a modifiedendogenous EPSPS gene encoding a plant EPSPS polypeptide that has theamino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6.

Methods for producing glyphosate tolerant plants are provided herein inwhich a guide RNA, one or more polynucleotide modification templates,and one or more Cas endonucleases are provided to a plant cell. The Casendonuclease(s) introduces a double strand break at an endogenous EPSPSgene in the plant cell, and the polynucleotide modification template(s)is used to generate a modified EPSPS gene that encodes a plant EPSPSpolypeptide that comprises G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2. A plant is obtained from the plant cell, and a glyphosatetolerant progeny plant that is void of the guide RNA and Casendonuclease is generated. In some embodiments, the one or morepolynucleotide modification templates are used to generate a modifiedendogenous EPSPS gene encoding a plant EPSPS polypeptide that comprisesG102A and at least two, at least three, or at least four amino acidmutations selected from the group consisting of: (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid mutation position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2.

In other embodiments, the one or more polynucleotide modificationtemplates are used to generate a modified endogenous EPSPS gene encodinga plant EPSPS polypeptide that comprises: A4W, H54M, L98C, G102A, K173R,I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q,and F442V. In still other embodiments, the one or more polynucleotidemodification templates are used to generate a modified endogenous EPSPSgene encoding a plant EPSPS polypeptide that comprises: A2R, A4W, A72Q,K84R, L98C, G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G,V438R, and T441Q. In still other embodiments, the one or morepolynucleotide modification templates are used to generate a modifiedendogenous EPSPS gene encoding a plant EPSPS polypeptide that comprises:A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, and D402G. In stillother embodiments, the one or more polynucleotide modification templatesare used to generate a modified endogenous EPSPS gene encoding a plantEPSPS polypeptide that has the amino acid sequence set forth in SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6.

Also provided herein are glyphosate tolerant maize plants that expressan endogenous EPSPS polypeptide that has G102A and at least one aminoacid mutation selected from the group consisting of: a) A2R, (b) A4W,(c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E,(j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p)A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2. A glyphosate tolerant maize plant may express a plant EPSPSpolypeptide having the sequence set forth in SEQ ID NO:4, SEQ ID NO:5,or SEQ ID NO:6.

Also provided herein are glyphosate tolerant sunflower plants thatexpress an EPSPS polypeptide that has G102A and at least one amino acidmutation selected from the group consisting of: a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:9. Aglyphosate tolerant sunflower plant may express a plant EPSPSpolypeptide having the sequence set forth in SEQ ID NO:10.

Also provided herein are glyphosate tolerant rice plants that express anEPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:7. Aglyphosate tolerant rice plant may express a plant EPSPS polypeptidehaving the sequence set forth in SEQ ID NO:11.

Also provided herein are glyphosate tolerant sorghum plants that expressan EPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:8. Aglyphosate tolerant sorghum plant may express a plant EPSPS polypeptidehaving the sequence set forth in SEQ ID NO:12.

Also provided herein are glyphosate tolerant soybean plants that expressan EPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:13. Aglyphosate tolerant soybean plant may express a plant EPSPS polypeptidehaving the sequence set forth in SEQ ID NO:18. Also provided herein areglyphosate tolerant wheat plants that express an EPSPS polypeptide thathas G102A and at least one amino acid mutation selected from the groupconsisting of: a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C,(g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m)E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q,and (t) F442V, wherein each amino acid position corresponds to theanalogous amino acid position set forth in SEQ ID NO:1 and wherein theplant EPSPS gene encodes a polypeptide comprising a sequence that atleast 90% identical to SEQ ID NO:14. A glyphosate tolerant wheat plantmay express a plant EPSPS polypeptide having the sequence set forth inSEQ ID NO:19.

Also provided herein are glyphosate tolerant Brassica rapa plants thatexpress an EPSPS polypeptide that has G102A and at least one amino acidmutation selected from the group consisting of: a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:15. Aglyphosate tolerant Brassica rapa plant may express a plant EPSPSpolypeptide having the sequence set forth in SEQ ID NO:20.

Also provided herein are glyphosate tolerant tomato plants that expressan EPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:17. Aglyphosate tolerant tomato plant may express a plant EPSPS polypeptidehaving the sequence set forth in SEQ ID NO:21.

Also provided herein are glyphosate tolerant potato plants that expressan EPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:18. Aglyphosate tolerant potato plant may express a plant EPSPS polypeptidehaving the sequence set forth in SEQ ID NO:22.

Methods of weed control in which an effective amount of glyphosate isapplied over a population of glyphosate tolerant plants provided hereinare also provided. The plants may be maize, sunflower, rice, wheat,tomato, potato, oil seed rape, sorghum, or soy. The effective amount ofglyphosate applied may be about 50 gram acid equivalent/acre to about2000 gram acid equivalent/acre.

Polynucleotide modification templates comprising a partial EPSP synthase(EPSPS) sequence, wherein a polynucleotide modification templatecomprises one or more nucleotide mutations that correspond to G102A andto at least one or more amino acid mutations selected from the groupconsisting of: a) A2R, b) A4W, c) H54M, d) A72Q, e) K84R, f) L98C, g)K173R, h) I208L, i) K243E, j) T279A, k) E302S, l) T361S, m) E391P, n)E391G, o) D402G, p) A416G, q) V438R, r) S440R, s) T441Q, and t) F442V,wherein each amino acid mutation position corresponds to the amino acidposition set forth in SEQ ID NO: 1, are also provided. Plant cellscomprising a polynucleotide modification template presented herein, aguide RNA, and CRISPR/Cas9 endonuclease are also provided wherein saidcombination targets an endogenous maize EPSPS sequence that encodes anEPSPS polypeptide that is at least 90% identical to SEQ ID NO:2.

Also provided is a method of rapidly assaying catalytic efficiency of aplurality of enzyme variants in the presence of an inhibitor. The methodincludes (a) providing a plurality of enzyme variants; (b) providing theinhibitor; (c) providing the substrate; (d) performing a reactioninvolving the plurality of enzyme variants and the substrate, at no morethan two different inhibitor concentrations; (e) measuring reaction rateat no more than two different inhibitor concentrations; and (f)calculating (kcat/KM)*KI of the plurality of enzyme variants. In someembodiments, one of the inhibitor concentrations is zero. In otherembodiments, the substrate is at a concentration that is substantiallysimilar to Michaelis-Menten constant (KM) of a parental enzyme for theenzyme variant. In still other embodiments, the enzyme is at asufficient concentration to result in a substantially linear reactionrate at the two different inhibitor concentrations. In still otherembodiments, one of the inhibitor concentrations is sufficient to resultin at least about 50% inhibition. In still other embodiments, the assayis performed in a high-throughput system. In still other embodiments,the catalytic capacity in the presence of the inhibitor is estimated byobtaining a numerical value for (kcat/KM)*KI, wherein kcat is maximumenzyme turnover rate, KM is Michaelis-Menten constant and KI isinhibitor dissociation constant. In some embodiments, the substrate isPEP; the inhibitor is glyphosate; and the plurality of enzyme variantsare EPSPS enzyme variants. In some embodiments, the enzyme and thesubstrate concentrations are the same, at the two inhibitorconcentrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of maize wild-type EPSPS amino acid sequence(SEQ ID NO:1) and a mutated H6 version (SEQ ID NO:5) of the EPSPSsequence.

FIG. 2 shows an alignment of soybean wild-type EPSPS amino acid sequence(SEQ ID NO:13) and a mutated H6 version (SEQ ID NO:18) of the EPSPSsequence.

FIG. 3 shows an alignment of sunflower wild-type EPSPS amino acidsequence (SEQ ID NO:9) and a mutated H6 version (SEQ ID NO:10) of theEPSPS sequence

FIG. 4 shows an alignment of rice wild-type EPSPS amino acid sequence(SEQ ID NO:7) and a mutated H6 version (SEQ ID NO:11) of the EPSPSsequence.

FIG. 5 shows an alignment of sorghum wild-type EPSPS amino acid sequence(SEQ ID NO:8) and a mutated H6 version (SEQ ID NO:12) of the EPSPSsequence.

FIG. 6 shows an alignment of wheat wild-type EPSPS amino acid sequence(SEQ ID NO:14) and a mutated H6 version (SEQ ID NO:19) of the EPSPSsequence.

FIG. 7 shows an alignment of B. rapa wild-type EPSPS amino acid sequence(SEQ ID NO:15) and a mutated H6 version (SEQ ID NO:20) of the EPSPSsequence.

FIG. 8 shows an alignment of Sorghum wild-type EPSPS amino acid sequence(SEQ ID NO:8) and a mutated C1 version (SEQ ID NO:23) of the EPSPSsequence.

FIG. 9 shows the growth of hairy roots from soybean cotyledonstransformed with native maize EPSPS or one of the shuffled variants.

FIG. 10 shows values of (k_(cat)/K_(M))*K_(I) determined by the rapidmethod compared with substrate saturation analysis. Data from Table 15are plotted as a linear regression. The values used for the rapid methodwere those obtained under adjusted conditions.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. § 1.8211.825. The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC IUBMB standards described inNucleic Acids Res. 13:3021 3030 (1985) and in the Biochemical J. 219(2):345 373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO:1 is the amino acid sequence of an expressed protein obtainedby cloning a synthetic EPSP synthase (in which the nucleotide sequenceof the gene encoding SEQ ID NO:2 was modified to add an N-terminalmethionine to SEQ ID NO:2 and to optimize codon usage for its expressionin E. coli) into an expression vector. SEQ ID NO:1 is to be used hereinas a reference EPSPS sequence.

SEQ ID NO:2 is the amino acid sequence of a maize EPSPS presented asGenBank entry CAA44974.1 (NCBI GI No. 1524383).

SEQ ID NO:3 is the amino acid sequence of the N-terminal extension.

SEQ ID NO:4 is the amino acid sequence of a maize EPSPS encoded by thenucleotide sequence present in clone 771-C2.

SEQ ID NO:5 is the amino acid sequence of a maize EPSPS encoded by thenucleotide sequence present in clone 868-H6.

SEQ ID NO:6 is the amino acid sequence of a maize EPSPS encoded by thenucleotide sequence present in clone 123-C1.

SEQ ID NO:7 is the amino acid sequence of a native EPSPS from rice(Oryza sativa) including the chloroplast transit peptide sequence.

SEQ ID NO:8 is the amino acid sequence of a native EPSPS from sorghum(Sorghum halepense) including the chloroplast transit peptide sequence.

SEQ ID NO:9 is an annotated amino acid sequence of a native EPSPS fromsunflower (Helianthus annus) including the chloroplast transit peptidesequence.

SEQ ID NO:10 is a mutated version of the EPSPS sequence from sunflower(SEQ ID NO:9) that contains the 868-H6 mutations.

SEQ ID NO:11 is a mutated version of the EPSPS sequence from rice (SEQID NO:7) that contains the 868-H6 mutations.

SEQ ID NO:12 is a mutated version of the EPSPS sequence from sorghum(SEQ ID NO:8) that contains the 868-H6 mutations.

SEQ ID NO:13 is an amino acid sequence of a native EPSPS from soybean(Glycine max) including the chloroplast transit peptide sequence.

SEQ ID NO:14 is an amino acid sequence of a native EPSPS from wheat(Triticum aestivum) including the chloroplast transit peptide sequence.

SEQ ID NO:15 is an amino acid sequence of a native EPSPS from Brassicarapa including the chloroplast transit peptide sequence.

SEQ ID NO:16 is an amino acid sequence of a native EPSPS from tomato(Solanum lycopersicum) including the chloroplast transit peptidesequence.

SEQ ID NO:17 is an amino acid sequence of a native EPSPS from potato(Solanum tuberosum) including the chloroplast transit peptide sequence.

SEQ ID NO:18 is a mutated version of the EPSPS sequence from soybean(SEQ ID NO:13) that contains the 868-H6 mutations.

SEQ ID NO:19 is a mutated version of the EPSPS sequence from wheat (SEQID NO:14) that contains the 868-H6 mutations.

SEQ ID NO:20 is a mutated version of the EPSPS sequence from Brassicarapa (SEQ ID NO:15) that contains the 868-H6 mutations.

SEQ ID NO:21 is a mutated version of the EPSPS sequence from tomato (SEQID NO:16) that contains the 868-H6 mutations.

SEQ ID NO:22 is a mutated version of the EPSPS sequence from potato

(SEQ ID NO:17) that contains the 868-H6 mutations.

SEQ ID NO:23 is a mutated version of the EPSPS sequence from sorghum(SEQ ID NO:8) that contains the 123-C1 mutations.

SEQ ID NO:24 is the DNA sequence that encodes the native maize EPSPS andthe C-terminal hemagglutinin affinity tag (but not the chloroplasttransit peptide).

SEQ ID NO:25 is the DNA sequence that encodes maize EPSPS variant 868-H6and the C-terminal hemagllutinin affinity tag (but not the chloroplasttransit peptide).

SEQ ID NO:26 is the DNA sequence coding for the chloroplast targetingpeptide from the Arabidopsis EPSPS.

SEQ ID NO:27 is the nucleotide sequence encoding an artificial CTPtermed 6H1 (U.S. Pat. No. 7,345,143).

SEQ ID NO:28 is the nucleotide sequence of the native Arabidopsis EPSPSpromoter (AT1G48860; NCBI GI No. CP002684.1, Arabidopsis thalianachromosome 1, base pairs 18071332 to Ser. No. 18/072,324).

SEQ ID NO:29 is the nucleotide sequence of the Arabidopsis thalianaubiquitin-3 promoter (NCBI GI No. GenBank L05363.1).

SEQ ID NO:30 is the nucleotide sequence of the promoter of theArabidopsis thaliana ubiquitin-10 (UBQ10) promoter.

SEQ ID NO:31 is the sequence of polynucleotide coding for thehemagglutinin affinity tag.

SEQ ID NO:32 is the nucleotide sequence of a phaseolin terminator.

SEQ ID NO:33 is the DNA sequence encoding the maize EPSPS variant C1 andthe C-terminal hemagglutinin affinity tag (but not the chloroplasttransit peptide).

SEQ ID NO:34 is the DNA sequence encoding the maize EPSPS variant C2 andthe C-terminal hemagglutinin affinity tag (but not the chloroplasttransit peptide).

DETAILED DESCRIPTION I. Compositions A. EPSP Synthase Polynucleotidesand Polypeptides

Various methods and compositions are provided which employpolynucleotides and polypeptides having EPSP synthase (EPSPS) activity.Such EPSPS polypeptides include those that encode plant EPSPSpolypeptides that comprise G102A and at least one or more amino acidmutations selected from the group consisting of: (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the plant EPSPS polypeptide comprises a sequence that is atleast 90% identical to SEQ ID NO:2.

In some embodiments, the polynucleotides encode plant EPSPS polypeptidesthat comprise G102A and at least two or more, three or more, or four ormore amino acid mutations selected from the group consisting of (a) A2R,(b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L,(i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o)D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid mutation position corresponds to the amino acidposition set forth in SEQ ID NO:1 and wherein the plant EPSPSpolypeptide comprises a sequence that is at least 90% identical to SEQID NO:2.

In other embodiments, the polynucleotide encodes a plant EPSPSpolypeptide that comprises A4W, H54M, L98C, G102A, K173R, I208L, K243E,E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q, and F442V (themutations present in clone 771-C2). In still other embodiments, thepolynucleotide encodes a plant EPSPS polypeptide that comprises A2R,A4W, A72Q, K84R, L98C, G102A, I208L, T279A, E302S, T361S, E391G, D402G,A416G, V438R, and T441Q (the mutations present in clone 868-H6). Instill other embodiments, the polynucleotide encodes a plant EPSPSpolypeptide that comprises A2R, A4W, K84R, L98C, G102A, I208L, K243E,E391P, and D402G (the mutations present in clone 123-C1). In still otherembodiments, the polynucleotide encodes the plant EPSPS polypeptide setforth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 which represent theamino acid sequences of maize EPSPS polypeptides encoded by thenucleotide sequences present in 771-C2, 868-H6, and 123-C1,respectively.

The EPSPS polypeptides and active variants and fragments thereofdisclosed herein may have improved catalytic capacity in the presence ofglyphosate when compared to previously identified EPSPS polypeptides.The parameter that best indicates the fitness of this trait in vivo isk_(cat)/K_(M)*K_(I). The EPSPS polypeptides disclosed herein can have anincreased k_(cat)/K_(M)*K_(I), when compared to previously known EPSPSenzymes. By “increase” is intended any statistically significantincrease when compared to an appropriate control. In some embodiments,an appropriate control is a previously known EPSPS sequence, such asthat set forth in SEQ ID NO:2 (maize), SEQ ID NO:7 (rice), SEQ ID NO:8(sorghum), SEQ ID NO:9 (sunflower), SEQ ID NO:13 (soybean), SEQ ID NO:14(wheat), SEQ ID NO:15 (Brassica rapa), SEQ ID NO:16 (tomato), or SEQ IDNO:17 (potato). In some embodiments, the increase in thek_(cat)/K_(M)*K_(I) when compared to SEQ ID NO:2, 7, 8 9, 13, 14, 15,16, or 17 can comprise about a 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800-fold or greater increase. In still furtherembodiments, k_(cat)/K_(M)*K_(I) may include, for example, ak_(cat)/K_(M)*K_(I) of more than about 2000, 2500, 3000, 3500, 4000,4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000,or more. The k_(cat)/K_(M)*K_(I) for the wild-type maize EPSPS is 11.8,while the k_(cat)/K_(M)*K_(I) of an EPSPS enzyme comprising 103I, 107S,and 445G is 2254.

As used herein, an “isolated” or “purified” polynucleotide orpolypeptide, or biologically active portion thereof, is substantially oressentially free from components that normally accompany or interactwith the polynucleotide or polypeptide as found in its naturallyoccurring environment. Thus, an isolated or purified polynucleotide orpolypeptide is substantially free of other cellular material or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Optimally, an “isolated” polynucleotide is free of sequences (optimallyprotein encoding sequences) that naturally flank the polynucleotide(i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) inthe genomic DNA of the organism from which the polynucleotide isderived. For purposes of this disclosure, “isolated” or “recombinant”when used to refer to nucleic acid molecules excludes isolatedunmodified chromosomes. For example, in various embodiments, theisolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flankthe polynucleotide in genomic DNA of the cell from which thepolynucleotide is derived. A polypeptide that is substantially free ofcellular material includes preparations of polypeptides having less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein.When the polypeptide of the disclosure or a biologically active portionthereof is recombinantly produced, optimally culture medium representsless than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemicalprecursors or non-protein-of-interest chemicals.

As used herein, a “recombinant” polynucleotide comprises a combinationof two or more chemically linked nucleic acid segments which are notfound directly joined in nature. By “directly joined” is intended thetwo nucleic acid segments are immediately adjacent and joined to oneanother by a chemical linkage. In specific embodiments, the recombinantpolynucleotide comprises a polynucleotide of interest or active variantor fragment thereof such that an additional chemically linked nucleicacid segment is located either 5′, 3′ or internal to the polynucleotideof interest. Alternatively, the chemically-linked nucleic acid segmentof the recombinant polynucleotide can be formed by the deletion of asequence. The additional chemically linked nucleic acid segment or thesequence deleted to join the linked nucleic acid segments can be of anylength, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 orgreater nucleotides. Various methods for making such recombinantpolynucleotides are disclosed herein, including, for example, bychemical synthesis or by the manipulation of isolated segments ofpolynucleotides by genetic engineering techniques. In specificembodiments, the recombinant polynucleotide can comprise a recombinantDNA sequence or a recombinant RNA sequence.

A “recombinant polypeptide” comprises a combination of two or morechemically linked amino acid segments which are not found directlyjoined in nature. In specific embodiments, the recombinant polypeptidecomprises an additional chemically linked amino acid segment that islocated either at the N-terminal, C-terminal or internal to therecombinant polypeptide. Alternatively, the chemically-linked amino acidsegment of the recombinant polypeptide can be formed by deletion of atleast one amino acid. The additional chemically linked amino acidsegment or the deleted chemically linked amino acid segment can be ofany length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20or amino acids.

B. Active Fragments and Variants of EPSPS Sequences

Methods and compositions are provided which employ polynucleotides andpolypeptides having EPSPS activity. Moreover, any given variant orfragment of an EPSPS sequence may further comprise an improved catalyticcapacity in the presence of the inhibitor glyphosate when compared to anappropriate control.

i. Polynucleotide and Polypeptide Fragments

Fragments and variants of the EPSPS polynucleotides and polypeptidesprovided herein are also encompassed by the present disclosure. By“fragment” is intended a portion of the polynucleotide or a portion ofthe amino acid sequence and hence protein encoded thereby. Fragments ofa polynucleotide may encode protein fragments that retain EPSPSactivity, and in specific embodiments, can further comprise an improvedproperty such as improved catalytic capacity in the presence ofglyphosate. Alternatively, fragments of a polynucleotide that are usefulas hybridization probes or PCR primers generally do not encode fragmentproteins retaining biological activity. In specific embodiments, afragment of a recombinant polynucleotide or a recombinant polynucleotideconstruct comprises at least one junction of the two or more chemicallylinked or operably linked nucleic acid segments which are not founddirectly joined in nature. Thus, fragments of a nucleotide sequence mayrange from at least about 20 nucleotides, about 50 nucleotides, about100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400nucleotides, about 500 nucleotides, about 600 nucleotides, about 700nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000nucleotides, about 1100 nucleotides, about 1200 nucleotides, about 1300nucleotides, and up to the full-length polynucleotide encoding the EPSPSpolypeptides. A fragment of an EPSPS polynucleotide that encodes abiologically active portion of an EPSPS protein of the disclosure willencode at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300,325, 350, 375, 400, or 425 amino acids, or up to the total number ofamino acids present in a full-length EPSPS polypeptide.

Thus, a fragment of an EPSPS polynucleotide may encode a biologicallyactive portion of an EPSPS polypeptide, or it may be a fragment that canbe used as a hybridization probe or PCR primer using methods disclosedbelow. A biologically active portion of an EPSPS polypeptide can beprepared by isolating a portion of one of the EPSPS polynucleotides,expressing the encoded portion of the EPSPS polypeptides (e.g., byrecombinant expression in vitro), and assessing the activity of theEPSPS portion of the EPSPS protein. Polynucleotides that are fragmentsof a EPSPS nucleotide sequence comprise at least 20, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 contiguousnucleotides, or up to the number of nucleotides present in a full-lengthEPSPS polynucleotide disclosed herein.

Fragments of a polypeptide may encode protein fragments that retainEPSPS activity, and in specific embodiments, can further comprise animproved catalytic capacity in the presence of glyphosate when comparedto an appropriate control. A fragment of a EPSPS polypeptide disclosedherein will encode at least 25, 50, 75, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, or 425 contiguous amino acids, or upto the total number of amino acids present in a full-length EPSPSpolypeptide. In specific embodiments, such polypeptide fragments areactive fragments, and in still other embodiments, the polypeptidefragment comprises a recombinant polypeptide fragment. As used herein, afragment of a recombinant polypeptide comprises at least one of acombination of two or more chemically linked amino acid segments whichare not found directly joined in nature.ii. Polynucleotide and Polypeptide Variants

“Variant” protein is intended to mean a protein derived from the proteinby deletion (i.e., truncation at the 5′ and/or 3′ end) and/or a deletionor addition of one or more amino acids at one or more internal sites inthe native protein and/or substitution of one or more amino acids at oneor more sites in the native protein. Variant proteins encompassed arebiologically active, that is they continue to possess the desiredbiological activity, that is, have EPSPS activity. Moreover, any givenvariant or fragment may further comprise an improved specificity forglyphosate when compared to an appropriate control resulting indecreased non-specific acetylation of, e.g. an amino acid such asaspartate. Such variants may result from, for example, geneticpolymorphism or from human manipulation.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having a deletion(i.e., truncations) at the 5′ and/or 3′ end and/or a deletion and/oraddition of one or more nucleotides at one or more internal sites withinthe native polynucleotide and/or a substitution of one or morenucleotides at one or more sites in the native polynucleotide. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the EPSPS polypeptides provided herein. Naturallyoccurring variants such as these can be identified with the use ofwell-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis or gene synthesis but which still encode anEPSPS polypeptide.

Biologically active variants of an EPSPS polypeptide disclosed herein(and the polynucleotide encoding the same) will have at least about 85%,90%, 91%₇ 92%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%,97.5%, 98%, 98.5%, 99%, 99.5%, or more sequence identity to thepolypeptide of any one of SEQ ID NO:1, 2, 4, 5, 6, 7, 8, 9, and 10, asdetermined by sequence alignment programs and parameters describedelsewhere herein.

The EPSPS polypeptide and the active variants and fragments thereof maybe altered in various ways including amino acid substitutions,deletions, truncations, and insertions. Methods for such manipulationsare generally known in the art. For example, amino acid sequencevariants and fragments of the EPSPS proteins can be prepared bymutations in the DNA. Methods for mutagenesis and polynucleotidealterations are well known in the art. See, for example, Kunkel (1985)Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods inEnzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.(1983) Techniques in Molecular Biology (MacMillan Publishing Company,New York) and the references cited therein. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be optimal.

The mutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

C. Sequence Comparisons

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides.

As used herein, “reference sequence” is a predetermined sequence used asa basis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene sequenceor protein sequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polypeptide sequence, wherein the polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two polypeptides.Generally, the comparison window is at least 5, 10, 15, or 20 contiguousamino acids in length, or it can be 30, 40, 50, 100, or longer. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polypeptide sequencea gap penalty is typically introduced and is subtracted from the numberof matches.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, or PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTP forproteins) can be used. Alignment may also be performed manually byinspection.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

D. Plants and Other Host Cells of Interest

Further provided are engineered host cells that are transduced(transformed or transfected) with one or more EPSPS sequences or activevariants or fragments thereof. The EPSPS polypeptides or variants andfragments thereof can be expressed in any organism, including innon-animal cells such as plants, yeast, fungi, bacteria and the like.Details regarding non-animal cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems, John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin, Heidelberg, New York); and Atlas and Parks(eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla.

Plants, plant cells, plant parts and seeds, and grain having the EPSPSsequences disclosed herein are also provided. In specific embodiments,the plants and/or plant parts have stably incorporated at least oneheterologous EPSPS polypeptide disclosed herein or an active variant orfragment thereof. In addition, the plants or organism of interest cancomprise multiple EPSPS polynucleotides (i.e., at least 1, 2, 3, 4, 5, 6or more).

In specific embodiments, the heterologous plant EPSPS polynucleotide inthe plant or plant part is operably linked to a heterologous regulatoryelement, such as but not limited to a constitutive, tissue-preferred, orother promoter for expression in plants or a constitutive enhancer.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the disclosure,provided that these parts comprise the introduced polynucleotides.

The EPSPS sequences and active variants and fragments thereof disclosedherein may be used for transformation of any plant species, including,but not limited to, monocots and dicots. Examples of plant species ofinterest include, but are not limited to, corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables, ornamentals, conifers, turf grasses (including cool seasonalgrasses and warm seasonal grasses).

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing that which is disclosedinclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specificembodiments, plants of the present disclosure are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments,corn and soybean plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e. with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

Additional host cells of interest can be a eukaryotic cell, an animalcell, a protoplast, a tissue culture cell, prokaryotic cell, a bacterialcell, such as E. coli, B. subtilis, Streptomyces, Salmonellatyphimurium, a gram positive bacteria, a purple bacteria, a green sulfurbacteria, a green non-sulfur bacteria, a cyanobacteria, a spirochetes, athermatogale, a flavobacteria, bacteroides; a fungal cell, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; aninsect cell such as Drosophila and Spodoptera frugiperda; a mammaliancell such as CHO, COS, BHK, HEK 293 or Bowes melanoma, archaebacteria(i.e., Korarchaeota, Thermoproteus, Pyrodictium, Thermococcales,Methanogens, Archaeoglobus, and extreme Halophiles) and others.

For example, in some embodiments, glyphosate tolerant maize plants areprovided, in which the glyphosate tolerant maize plants express anendogenous EPSPS polypeptide that has G102A and at least one amino acidmutation selected from the group consisting of: a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the endogenous plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:2.Further, the glyphosate tolerant maize plant may express an endogenousEPSPS polypeptide that has G102A and at least two, at least three, or atleast four of the amino acid mutations selected from the groupconsisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C,(g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m)E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q,and (t) F442V, wherein each amino acid position corresponds to the aminoacid position set forth in SEQ ID NO:1 and wherein the endogenous plantEPSPS gene encodes a polypeptide comprising a sequence that is at least90% identical to SEQ ID NO:2. Still further, the glyphosate tolerantmaize plant may express an endogenous EPSPS polypeptide that has: (a)A4W, H54M, L98C, G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G,A416G, V438R, S440R, T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C,G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G, V438R, andT441Q; or (c) A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, andD402G; or has the sequence set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQID NO:6.

For example, in some embodiments, glyphosate tolerant sunflower plantsare provided, in which the glyphosate tolerant sunflower plants expressan EPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the plant EPSPS gene encodes a polypeptide comprising asequence that is at least 90% identical to SEQ ID NO:9. Further, theglyphosate tolerant sunflower plant may express an EPSPS polypeptidethat has G102A and at least two, at least three, or at least four of theamino acid mutations selected from the group consisting of: (a) A2R, (b)A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i)K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G,(p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:9.Still further, the glyphosate tolerant sunflower plant may express anendogenous EPSPS polypeptide that has: (a) A4W, H54M, L98C, G102A,K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W,K84R, L98C, G102A, I208L, K243E, E391P, and D402G. A glyphosate tolerantsunflower plant may express an EPSPS polypeptide having the sequence setforth in SEQ ID NO:10.

For example, in some embodiments, glyphosate tolerant rice plants areprovided, in which the glyphosate tolerant rice plants express an EPSPSpolypeptide that has G102A and at least one amino acid mutation selectedfrom the group consisting of: a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e)K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S,(l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r)S440R, (s) T441Q, and (t) F442V, wherein each amino acid positioncorresponds to the amino acid position set forth in SEQ ID NO:1 andwherein the plant EPSPS gene encodes a polypeptide comprising a sequencethat is at least 90% identical to SEQ ID NO:7. Further, the glyphosatetolerant rice plant may express an EPSPS polypeptide that has G102A andat least two, at least three, or at least four of the amino acidmutations selected from the group consisting of: (a) A2R, (b) A4W, (c)H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j)T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G,(q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the plant EPSPS gene encodes a polypeptide comprising asequence that is at least 90% identical to SEQ ID NO:7. Still further,the glyphosate tolerant rice plant may express an EPSPS polypeptide thathas: (a) A4W, H54M, L98C, G102A, K173R, I208L, K243E, E302S, T361S,E391P, D402G, A416G, V438R, S440R, T441Q, and F442V; (b) A2R, A4W, A72Q,K84R, L98C, G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G,V438R, and T441Q; or (c) A2R, A4W, K84R, L98C, G102A, I208L, K243E,E391P, and D402G. A glyphosate tolerant rice plant may express the EPSPSpolypeptide set forth in SEQ ID NO:11.

For example, in some embodiments, glyphosate tolerant sorghum plants areprovided, in which the glyphosate tolerant sorghum plants express anEPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the amino acid position set forth in SEQ ID NO:1and wherein the plant EPSPS gene encodes a polypeptide comprising asequence that is at least 90% identical to SEQ ID NO:8. Further, theglyphosate tolerant sorghum plant may express an EPSPS polypeptide thathas G102A and at least two, at least three, or at least four of theamino acid mutations selected from the group consisting of: (a) A2R, (b)A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i)K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G,(p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:8.Still further, the glyphosate tolerant sorghum plant may express anEPSPS polypeptide that has: (a) A4W, H54M, L98C, G102A, K173R, I208L,K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q, andF442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A, E302S,T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W, K84R,L98C, G102A, I208L, K243E, E391P, and D402G. A glyphosate tolerantsorghum plant may express an EPSPS polypeptide having the sequence setforth in SEQ ID NO:12.

For example, in some embodiments, glyphosate tolerant soybean plants areprovided, in which the glyphosate tolerant soybean plants express anEPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:13.Further, the glyphosate tolerant soybean plant may express an EPSPSpolypeptide that has G102A and at least two, at least three, or at leastfour of the amino acid mutations selected from the group consisting of:(a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h)I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G,(o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid position corresponds to the amino acid positionset forth in SEQ ID NO:1 and wherein the plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:13. Still further, the glyphosate tolerant soybean plant mayexpress an EPSPS polypeptide that has: (a) A4W, H54M, L98C, G102A,K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W,K84R, L98C, G102A, I208L, K243E, E391P, and D402G. A glyphosate tolerantsoybean plant may express a plant EPSPS polypeptide having the sequenceset forth in SEQ ID NO:18. For example, in some embodiments, glyphosatetolerant wheat plants are provided, in which the glyphosate tolerantwheat plants express an EPSPS polypeptide that has G102A and at leastone amino acid mutation selected from the group consisting of: a) A2R,(b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L,(i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o)D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid position corresponds to the analogous amino acidposition set forth in SEQ ID NO:1 and wherein the plant EPSPS geneencodes a polypeptide comprising a sequence that is at least 90%identical to SEQ ID NO:14. Further, the glyphosate tolerant wheat plantmay express an EPSPS polypeptide that has G102A and at least two, atleast three, or at least four of the amino acid mutations selected fromthe group consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R,(f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l)T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R,(s) T441Q, and (t) F442V, wherein each amino acid position correspondsto the amino acid position set forth in SEQ ID NO:1 and wherein theplant EPSPS gene encodes a polypeptide comprising a sequence that is atleast 90% identical to SEQ ID NO:14. Still further, the glyphosatetolerant wheat plant may express an EPSPS polypeptide that has: (a) A4W,H54M, L98C, G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G,A416G, V438R, S440R, T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C,G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G, V438R, andT441Q; or (c) A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, andD402G. A glyphosate tolerant wheat plant may express a plant EPSPSpolypeptide having the sequence set forth in SEQ ID NO:19.

For example, in some embodiments, glyphosate tolerant Brassica rapaplants are provided, in which the glyphosate tolerant Brassica rapaplants express an EPSPS polypeptide that has G102A and at least oneamino acid mutation selected from the group consisting of: a) A2R, (b)A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i)K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G,(p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid position corresponds to the analogous amino acid position setforth in SEQ ID NO:1 and wherein the plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:15. Further, the glyphosate tolerant Brassica rapa plant mayexpress an EPSPS polypeptide that has G102A and at least two, at leastthree, or at least four of the amino acid mutations selected from thegroup consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f)L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S,(m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s)T441Q, and (t) F442V, wherein each amino acid position corresponds tothe amino acid position set forth in SEQ ID NO:1 and wherein the plantEPSPS gene encodes a polypeptide comprising a sequence that is at least90% identical to SEQ ID NO:15. Still further, the glyphosate tolerantBrassica rapa plant may express an EPSPS polypeptide that has: (a) A4W,H54M, L98C, G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G,A416G, V438R, S440R, T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C,G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G, V438R, andT441Q; or (c) A2R, A4W, K84R, L98C, G102A, I208L, K243E, E391P, andD402G. A glyphosate tolerant Brassica rapa plant may express a plantEPSPS polypeptide having the sequence set forth in SEQ ID NO:20.

For example, in some embodiments, glyphosate tolerant tomato plants areprovided, in which the glyphosate tolerant tomato plants express anEPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:17.Further, the glyphosate tolerant tomato plant may express an EPSPSpolypeptide that has G102A and at least two, at least three, or at leastfour of the amino acid mutations selected from the group consisting of:(a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h)I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G,(o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid position corresponds to the amino acid positionset forth in SEQ ID NO:1 and wherein the plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:17. Still further, the glyphosate tolerant tomato plant mayexpress an EPSPS polypeptide that has: (a) A4W, H54M, L98C, G102A,K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W,K84R, L98C, G102A, I208L, K243E, E391P, and D402G. A glyphosate toleranttomato plant may express a plant EPSPS polypeptide having the sequenceset forth in SEQ ID NO:21.

For example, in some embodiments, glyphosate tolerant potato plants areprovided, in which the glyphosate tolerant potato plants express anEPSPS polypeptide that has G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidposition corresponds to the analogous amino acid position set forth inSEQ ID NO:1 and wherein the plant EPSPS gene encodes a polypeptidecomprising a sequence that is at least 90% identical to SEQ ID NO:18.Further, the glyphosate tolerant potato plant may express an EPSPSpolypeptide that has G102A and at least two, at least three, or at leastfour of the amino acid mutations selected from the group consisting of:(a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h)I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G,(o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid position corresponds to the amino acid positionset forth in SEQ ID NO:1 and wherein the plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:18. Still further, the glyphosate tolerant potato plant mayexpress an EPSPS polypeptide that has: (a) A4W, H54M, L98C, G102A,K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W,K84R, L98C, G102A, I208L, K243E, E391P, and D402G. A glyphosate tolerantpotato plant may express a plant EPSPS polypeptide having the sequenceset forth in SEQ ID NO:22.

E. Polynucleotide Constructs

The use of the term “polynucleotide” is not intended to limit apolynucleotide of the disclosure to a polynucleotide comprising DNA.Those of ordinary skill in the art will recognize that polynucleotidescan comprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the disclosure also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

For example, a polynucleotide construct may be a recombinant DNAconstruct. A “recombinant DNA construct” comprises two or more operablylinked DNA segments which are not found operably linked in nature.Non-limiting examples of recombinant DNA constructs include apolynucleotide of interest or active variant or fragment thereofoperably linked to heterologous sequences which aid in the expression,autologous replication, and/or genomic insertion of the sequence ofinterest. Such heterologous and operably linked sequences include, forexample, promoters, termination sequences, enhancers, etc., or anycomponent of an expression cassette; a plasmid, cosmid, virus,autonomously replicating sequence, phage, or linear or circularsingle-stranded or double-stranded DNA or RNA nucleotide sequence;and/or sequences that encode heterologous polypeptides.

The EPSPS polynucleotides disclosed herein can be provided in expressioncassettes for expression in the plant of interest or any organism ofinterest. The cassette can include 5′ and 3′ regulatory sequencesoperably linked to an EPSPS polynucleotide or active variant or fragmentthereof. “Operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites and/or recombination sites for insertion of the EPSPSpolynucleotide or active variant or fragment thereof to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a EPSPS polynucleotide or active variant or fragmentthereof, and a transcriptional and translational termination region(i.e., termination region) functional in plants. The regulatory regions(i.e., promoters, transcriptional regulatory regions, and translationaltermination regions) and/or the EPSPS polynucleotide or active variantor fragment thereof may be native/analogous to the host cell or to eachother. Alternatively, the regulatory regions and/or the EPSPSpolynucleotide of or active variant or fragment thereof may beheterologous to the host cell or to each other.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

The termination region may be native with the transcriptional initiationregion or active variant or fragment thereof, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous) to the promoter, the EPSPS polynucleotide or activefragment or variant thereof, the plant host, or any combination thereof.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include viral translational leadersequences.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used to express the various EPSPS sequencesdisclosed herein, including the native promoter of the polynucleotidesequence of interest. The promoters can be selected based on the desiredoutcome. Such promoters include, for example, constitutive, inducible,tissue-preferred, or other promoters for expression in plants or in anyorganism of interest.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Synthetic promoters can be used to express EPSPS sequences orbiologically active variants and fragments thereof. Synthetic promotersinclude for example a combination of one or more heterologous regulatoryelements.

In another aspect, the EPSPS sequences disclosed herein or activevariants or fragments thereof can also be used as a selectable markergene. In this embodiment, the presence of the EPSPS polynucleotide in acell or organism confers upon the cell or organism the detectablephenotypic trait of glyphosate resistance, thereby allowing one toselect for cells or organisms that have been transformed with a gene ofinterest linked to the EPSPS polynucleotide. Thus, for example, theEPSPS polynucleotide can be introduced into a nucleic acid construct,e.g., a vector, thereby allowing for the identification of a host (e.g.,a cell or transgenic plant) containing the nucleic acid construct bygrowing the host in the presence of glyphosate and selecting for theability to survive and/or grow at a rate that is discernibly greaterthan a host lacking the nucleic acid construct would survive or grow. AnEPSPS polynucleotide can be used as a selectable marker in a widevariety of hosts that are sensitive to glyphosate, including plants,most bacteria (including E. coli), actinomycetes, yeasts, algae andfungi.

In specific embodiments, the EPSPS polypeptides and active variants andfragments thereof, and polynucleotides encoding the same, furthercomprise a chloroplast transit peptide. As used herein, the term“chloroplast transit peptide” will be abbreviated “CTP” and refers tothe N-terminal portion of a chloroplast precursor protein that directsthe latter into chloroplasts and is subsequently cleaved off by thechloroplast processing protease. When a CTP is operably linked to theN-terminus of a polypeptide, the polypeptide is translocated into thechloroplast. Removal of the CTP from a native protein reduces orabolishes the ability of the native protein from being transported intothe chloroplast. An operably linked chloroplast transit peptide is foundat the N-terminus of the protein to be targeted to the chloroplast andis located upstream and immediately adjacent to the transit peptidecleavage site that separates the transit peptide from the mature proteinto be targeted to the chloroplast.

The term “chloroplast transit peptide cleavage site” refers to a sitebetween two amino acids in a chloroplast-targeting sequence at which thechloroplast processing protease acts. Chloroplast transit peptidestarget the desired protein to the chloroplast and can facilitate theproteins translocation into the organelle. This is accompanied by thecleavage of the transit peptide from the mature polypeptide or proteinat the appropriate transit peptide cleavage site by a chloroplastprocessing protease, native to the chloroplast. Accordingly, achloroplast transit peptide further comprises a suitable cleavage sitefor the correct processing of the pre-protein to the mature polypeptidecontained within the chloroplast.

As used herein, a “heterologous” CTP comprises a transit peptidesequence which is foreign to the polypeptide it is operably linked to.Such heterologous chloroplast transit peptides are known, including butnot limited to those derived from Pisum (JP 1986224990; E00977), carrot(Luo et al. (1997) Plant Mol. Biol., 33 (4), 709-722 (Z33383), Nicotiana(Bowler et al., EP 0359617; A09029), Oryza (de Pater et al. (1990) PlantMol. Biol., 15 (3), 399-406 (X51911), as well as synthetic sequencessuch as those provided in EP 0189707; U.S. Pat. Nos. 5,728,925;5,717,084 (A10396 and A10398). In one embodiment, the heterologouschloroplast transit peptide is from the ribulose-1,5-bisphosphatecarboxylase (Rubisco) small subunit precursor protein isolated from anyplant. The Rubisco small subunit is well characterized from a variety ofplants and the transit peptide from any of them will be suitable for usedisclosed herein. See for example, Physcomitrella (Quatrano et al.,AW599738); Lotus (Poulsen et al., AW428760); Citrullus (J. S. Shin,A1563240); Nicotiana (Appleby et al. (1997) Heredity 79(6), 557-563);alfalfa (Khoudi et al. (1997) Gene, 197(1/2), 343-351); potato andtomato (Fritz et al. (1993) Gene, 137(2), 271-4); wheat (Galili et al.(1991) Theor. Appl. Genet. 81(1), 98-104); and rice (Xie et al. (1987)Sci. Sin., Ser. B (Engl. Ed.), 30(7), 706-19). For example, transitpeptides may be derived from the Rubisco small subunit isolated fromplants including but not limited to, soybean, rapeseed, sunflower,cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice,Arabidopsis, sugar beet, sugar cane, canola, millet, beans, peas, rye,flax, and forage grasses. Preferred for use in the present disclosure isthe Rubisco small subunit precursor protein from, for example,Arabidopsis or tobacco.

F. Stacking Other Traits of Interest

In some embodiments, the EPSPS polynucleotides or active variants andfragments thereof disclosed herein are engineered into a molecularstack. Thus, the various host cells, plants, plant cells and seedsdisclosed herein can further comprise one or more traits of interest,and in more specific embodiments, the host cell, plant, plant part orplant cell is stacked with any combination of polynucleotide sequencesof interest in order to create plants with a desired combination oftraits. As used herein, the term “stacked” includes having the multipletraits present in the same plant or organism of interest. In onenon-limiting example, “stacked traits” comprise a molecular stack wherethe sequences are physically adjacent to each other. A trait, as usedherein, refers to the phenotype derived from a particular sequence orgroups of sequences. In one embodiment, the molecular stack comprises atleast one additional polynucleotide that also confers tolerance to atleast one sequence that confers tolerance to glyphosate by the sameand/or different mechanism and/or at least one additional polynucleotidethat confers tolerance to a second herbicide.

Thus, in one embodiment, the host cells, plants, plant cells or plantpart having the EPSPS polynucleotide or active variants or fragmentsthereof disclosed herein is stacked with at least one other EPSPSsequence. Such EPSPS sequence include the EPSPS sequence and variantsand fragment thereof disclosed herein, as well as other EPSPS sequences,which include but are not limited to, the EPSPS sequences set forth inWO02/36782, US Publication 2004/0082770 and WO 2005/012515, U.S. Pat.Nos. 7,462,481, 7,405,074, each of which is herein incorporated byreference.

The mechanism of glyphosate tolerance produced by the EPSPS sequencesdisclosed herein may be combined with other modes of herbicideresistance to provide host cells, plants, plant explants and plant cellsthat are tolerant to glyphosate and one or more other herbicides. Forinstance, the mechanism of glyphosate tolerance conferred by EPSPS maybe combined with other modes of glyphosate tolerance known in the art.In other embodiments, the plant or plant cell or plant part having theEPSPS sequence or an active variant or fragment thereof may be stackedwith, for example, one or more sequences that confer tolerance to: anALS inhibitor; an HPPD inhibitor; 2,4-D; other phenoxy auxin herbicides;aryloxyphenoxypropionate herbicides; dicamba; glutamine synthetase (GS);glufosinate herbicides; herbicides which target the protox enzyme (alsoreferred to as “protox inhibitors”).

The plant or plant cell or plant part having the EPSPS sequence or anactive variant or fragment thereof can also be combined with at leastone other trait to produce plants that further comprise a variety ofdesired trait combinations. For instance, the plant or plant cell orplant part having the EPSPS sequence or an active variant or fragmentthereof may be stacked with polynucleotides encoding polypeptides havingpesticidal and/or insecticidal activity, or a plant or plant cell orplant part having the EPSPS sequence or an active variant or fragmentthereof may be combined with a plant disease resistance gene.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology, orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes (trans) or contained onthe same transformation cassette (cis). Expression of the sequences canbe driven by the same promoter or by different promoters. In certaincases, it may be desirable to introduce a transformation cassette thatwill suppress the expression of the polynucleotide of interest. This maybe combined with any combination of other suppression cassettes oroverexpression cassettes to generate the desired combination of traitsin the plant. It is further recognized that polynucleotide sequences canbe stacked at a desired genomic location using a site-specificrecombination system. See, for example, WO99/25821, WO99/25854,WO99/25840, WO99/25855, and WO99/25853, all of which are hereinincorporated by reference.

Any plant having at EPSPS sequence disclosed herein or an active variantor fragment thereof can be used to make a food or a feed product. Suchmethods comprise obtaining a plant, explant, seed, plant cell, or cellcomprising the EPSPS sequence or active variant or fragment thereof andprocessing the plant, explant, seed, plant cell, or cell to produce afood or feed product.

II. Methods of Use A. Methods of Generating Glyphosate Tolerant Plants

The terms “glyphosate tolerance” and “glyphosate resistance” are usedinterchangeably herein.

i. Introducing

Various methods can be used to introduce a sequence of interest into ahost cell, plant or plant part. “Introducing” is intended to meanpresenting to the host cell, plant, plant cell or plant part thepolynucleotide or polypeptide in such a manner that the sequence gainsaccess to the interior of a cell of the plant or organism. The methodsof the disclosure do not depend on a particular method for introducing asequence into an organism or a plant or plant part, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the organism or the plant. Methods for introducingpolynucleotide or polypeptides into various organisms, including plants,are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant or organism of interest and is capable of being inherited by theprogeny thereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the plant or organism of interest anddoes not integrate into the genome of the plant or organism or apolypeptide is introduced into a plant or organism.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballisticparticle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;5,879,918; 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell,Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology6:923-926); and Lec1 transformation (WO 00/28058). Also see Weissingeret al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990)Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al.(1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) PlantCell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the EPSPS sequences or active variants orfragments thereof can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, the introduction of the EPSPS proteinor active variants and fragments thereof directly into the plant. Suchmethods include, for example, microinjection or particle bombardment.See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185;Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc.Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal ofCell Science 107:775-784, all of which are herein incorporated byreference.

In other embodiments, the EPSPS polynucleotide disclosed herein oractive variants and fragments thereof may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating a nucleotide construct of the disclosurewithin a DNA or RNA molecule. It is recognized that the EPSPS sequencemay be initially synthesized as part of a viral polyprotein, which latermay be processed by proteolysis in vivo or in vitro to produce thedesired recombinant protein. Further, it is recognized that promotersdisclosed herein also encompass promoters utilized for transcription byviral RNA polymerases. Methods for introducing polynucleotides intoplants and expressing a protein encoded therein, involving viral DNA orRNA molecules, are known in the art. See, for example, U.S. Pat. Nos.5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al.(1996) Molecular Biotechnology 5:209-221; herein incorporated byreference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide disclosed herein can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome. Other methods to target polynucleotides are set forth inWO 2009/114321 (herein incorporated by reference), which describes“custom” meganucleases produced to modify plant genomes, in particularthe genome of maize. See, also, Gao et al. (2010) Plant Journal1:176-187.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present disclosure provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide disclosedherein, for example, as part of an expression cassette, stablyincorporated into their genome.

Transformed plant cells which are derived by plant transformationtechniques, including those discussed above, can be cultured toregenerate a whole plant which possesses the transformed genotype (i.e.,a EPSPS polynucleotide), and thus the desired phenotype, such asacquired resistance (i.e., tolerance) to glyphosate or a glyphosateanalog. For transformation and regeneration of maize see, Gordon-Kamm etal., The Plant Cell, 2:603-618 (1990). Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp 124-176, MacmillanPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can alsobe obtained from plant callus, explants, organs, or parts thereof. Suchregeneration techniques are described generally in Klee et al. (1987)Ann Rev of Plant Phys 38:467. See also, e.g., Payne and Gamborg.

One of skill will recognize that after the expression cassettecontaining the EPSPS gene is stably incorporated in transgenic plantsand confirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included, provided that theseparts comprise cells comprising the EPSPS nucleic acid. Progeny andvariants, and mutants of the regenerated plants are also included,provided that these parts comprise the introduced nucleic acidsequences.

In one embodiment, a homozygous transgenic plant can be obtained bysexually mating (selfing) a heterozygous transgenic plant that containsa single added heterologous nucleic acid, germinating some of the seedproduced and analyzing the resulting plants produced for altered celldivision relative to a control plant (i.e., native, non-transgenic).Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

Animal and lower eukaryotic (e.g., yeast) host cells are competent orrendered competent for transfection by various means. There are severalwell-known methods of introducing DNA into animal cells. These methodsinclude: calcium phosphate precipitation; fusion of the recipient cellswith bacterial protoplasts containing the DNA; treatment of therecipient cells with liposomes containing the DNA; DEAE dextran;electroporation; biolistics; and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. See, Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

In some embodiments, the methods comprise introducing by way ofexpressing in a regenerable plant cell a recombinant DNA constructcomprising a polynucleotide operably linked to at least one regulatorysequence, wherein the polynucleotide encodes a plant EPSPS polypeptidethat comprises G102A and at least one amino acid mutation selected fromthe group consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R,(f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l)T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R,(s) T441Q, and (t) F442V, wherein each amino acid mutation positioncorresponds to the amino acid position set forth in SEQ ID NO:1 andwherein the plant EPSPS polypeptide comprises a sequence that is atleast 90% identical to SEQ ID NO:2; and generating a glyphosate tolerantplant that comprises in its genome the recombinant DNA construct. Insome embodiments, the methods include expressing in a plant cell arecombinant DNA construct comprising a polynucleotide encoding a plantEPSPS polypeptide comprising G102A and at least two, at least three, orat least four amino acid mutations selected from the group consistingof: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R,(h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n)E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t)F442V, wherein each amino acid position corresponds to the amino acidposition set forth in SEQ ID NO:1 and wherein the plant EPSPSpolypeptide comprises a sequence that is at least 90% identical to SEQID NO:2.

The recombinant DNA may comprise: (a) a polynucleotide that encodes aplant EPSPS polypeptide that comprises A4W, H54M, L98C, G102A, K173R,I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R, T441Q,and F442V; (b) a polynucleotide that encodes a plant EPSPS polypeptidethat comprises A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A, E302S,T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) a polynucleotidethat encodes a plant EPSPS polypeptide that comprises A2R, A4W, K84R,L98C, G102A, I208L, K243E, E391P, and D402G. The recombinant DNA mayalso comprise a polynucleotide that encodes the plant EPSPS polypeptideset forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

ii. Modifying

In general, methods to modify or alter the host genomic DNA areavailable. For example, a pre-existing or endogenous EPSPS sequence in ahost plant can be modified or altered in a site-specific fashion usingone or more site-specific engineering systems. This includes alteringthe host DNA sequence or a pre-existing transgenic sequence includingregulatory elements, coding and non-coding sequences. These methods arealso useful in targeting nucleic acids to pre-engineered targetrecognition sequences in the genome. As an example, the geneticallymodified cell or plant described herein, is generated using “custom” orengineered endonucleases such as meganucleases produced to modify plantgenomes (see e.g., WO 2009/114321; Gao et al. (2010) Plant Journal1:176-187). Another site-directed engineering is through the use of zincfinger domain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The CRISPR/Cas system allows targeted cleavage of genomicDNA guided by a customizable small noncoding RNA.

For instance, an endogenous plant EPSPS gene in a plant cell may bemodified to encode a glyphosate tolerant EPSPS protein that comprisesG102A and at least one amino acid mutation selected from the groupconsisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C,(g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m)E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q,and (t) F442V, wherein each amino acid mutation position corresponds tothe amino acid position set forth in SEQ ID NO:1 and wherein theendogenous plant EPSPS gene encodes a polypeptide comprising a sequencethat is at least 90% identical to SEQ ID NO:2. A glyphosate tolerantplant may be grown from the plant cell. The modified endogenous plantEPSPS gene may encode a glyphosate tolerant EPSPS protein that comprisesG102A and at least two, at least three, or at least four of the aminoacid mutations selected from the group consisting of: (a) A2R, (b) A4W,(c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E,(j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p)A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2. The modified endogenous plant EPSPS gene may encode aglyphosate tolerant EPSPS protein that comprises: (a) A4W, H54M, L98C,G102A, K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R,S440R, T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L,T279A, E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R,A4W, K84R, L98C, G102A, I208L, K243E, E391P, and D402G. The modifiedendogenous plant EPSPS gene may encode a glyphosate tolerant EPSPSprotein that comprises the plant EPSPS polypeptide set forth in SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6.

The endogenous plant EPSPS gene may be modified by a CRISPR/Cas guideRNA-mediated system, a Zn-finger nuclease-mediated system, ameganuclease-mediated system, an oligonucleobase-mediated system, or anygene modification system known to one of ordinary skill in the art.

Moreover, for the purposes herein, an endogenous plant EPSPS geneincludes coding DNA and genomic DNA within and surrounding the codingDNA, such as for example, the promoter, intron, and terminatorsequences.

In some embodiments, the CRISPR/Cas guide RNA-mediated system is used tomodify the endogenous plant EPSPS gene. CRISPRs are arrays of clustered,regularly interspaced, short palindromic repeats within the bacterialgenome. The recent discovery of CRISPR-associated protein 9 nuclease(Cas9) from Streptococcus pyogenes presents the possibility ofintroducing mutations into a native gene (Sander and Joung, 2014). Tointroduce double strand breaks into the target gene, Cas9 is guided tothe target gene DNA by normal base-pairing with an engineered RNA.Following double-strand break, the desired mutation(s) in EPSPS can beintroduced from an engineered template through the homology-directedrepair process. EPSPS coded by modified genes will be under the controlof the native promoter. Thus, all tissues will express the enzymeaccording to their native spatial and temporal program, a condition thatmay confer an advantage over transgenic expression in providingappropriate catalytic capacity.

As used herein, the term “guide polynucleotide”, refers to apolynucleotide sequence that can form a complex with a Cas endonucleaseand enables the Cas endonuclease to recognize and optionally cleave aDNA target site. The guide polynucleotide can include a single moleculeor a double molecule. The guide polynucleotide sequence can be a RNAsequence, a DNA sequence, or a combination thereof (a RNA-DNAcombination sequence). Optionally, the guide polynucleotide can compriseat least one nucleotide, phosphodiester bond or linkage modificationsuch as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC,2,6-Diaminopurine, 2′-Fluoro A, 2′-Fluoro U, 2′-O-Methyl RNA,Phosphorothioate bond, linkage to a cholesterol molecule, linkage to apolyethylene glycol molecule, linkage to a spacer 18 (hexaethyleneglycol chain) molecule, or 5′ to 3′ covalent linkage resulting incircularization. In some embodiment of this disclosure, the guidepolynucleotide does not solely comprise ribonucleic acids (RNAs). Aguide polynucleotide that solely comprises ribonucleic acids is alsoreferred to as a “guide RNA”.

The guide polynucleotide can be a double molecule (also referred to asduplex guide polynucleotide) comprising a first nucleotide sequencedomain (referred to as Variable Targeting domain or VT domain) that iscomplementary to a nucleotide sequence in a target DNA and a secondnucleotide sequence domain (referred to as Cas endonuclease recognitiondomain or CER domain) that interacts with a Cas endonucleasepolypeptide. The CER domain of the double molecule guide polynucleotidecomprises two separate molecules that are hybridized along a region ofcomplementarity. The two separate molecules can be RNA, DNA, and/orRNA-DNA-combination sequences. In some embodiments, the first moleculeof the duplex guide polynucleotide comprising a VT domain linked to aCER domain is referred to as “crDNA” (when composed of a contiguousstretch of DNA nucleotides) or “crRNA” (when composed of a contiguousstretch of RNA nucleotides), or “crDNA-RNA” (when composed of acombination of DNA and RNA nucleotides). The crNucleotide can comprise afragment of the crRNA naturally occurring in Bacteria and Archaea. Inone embodiment, the size of the fragment of the crRNA naturallyoccurring in Bacteria and Archaea that is present in a crNucleotidedisclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.In some embodiments the second molecule of the duplex guidepolynucleotide comprising a CER domain is referred to as “tracrRNA”(when composed of a contiguous stretch of RNA nucleotides) or “tracrDNA”(when composed of a contiguous stretch of DNA nucleotides) or“tracrDNA-RNA” (when composed of a combination of DNA and RNAnucleotides In one embodiment, the RNA that guides the RNA/Cas9endonuclease complex, is a duplexed RNA comprising a duplexcrRNA-tracrRNA.

The guide polynucleotide can also be a single molecule comprising afirst nucleotide sequence domain (referred to as Variable Targetingdomain or VT domain) that is complementary to a nucleotide sequence in atarget DNA and a second nucleotide domain (referred to as Casendonuclease recognition domain or CER domain) that interacts with a Casendonuclease polypeptide. By “domain” it is meant a contiguous stretchof nucleotides that can be RNA, DNA, and/or RNA-DNA-combinationsequence. The VT domain and/or the CER domain of a single guidepolynucleotide can comprise a RNA sequence, a DNA sequence, or aRNA-DNA-combination sequence. In some embodiments the single guidepolynucleotide comprises a crNucleotide (comprising a VT domain linkedto a CER domain) linked to a tracrNucleotide (comprising a CER domain),wherein the linkage is a nucleotide sequence comprising a RNA sequence,a DNA sequence, or a RNA-DNA combination sequence. The single guidepolynucleotide being comprised of sequences from the crNucleotide andtracrNucleotide may be referred to as “single guide RNA” (when composedof a contiguous stretch of RNA nucleotides) or “single guide DNA” (whencomposed of a contiguous stretch of DNA nucleotides) or “single guideRNA-DNA” (when composed of a combination of RNA and DNA nucleotides).

In one embodiment of the disclosure, the single guide RNA comprises acrRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the typeII CRISPR/Cas system that can form a complex with a type II Casendonuclease, wherein said guide RNA/Cas endonuclease complex can directthe Cas endonuclease to a plant genomic target site, enabling the Casendonuclease to introduce a double strand break into the genomic targetsite.

One aspect of using a single guide polynucleotide versus a duplex guidepolynucleotide is that only one expression cassette needs to be made toexpress the single guide polynucleotide.

The term “variable targeting domain” or “VT domain” is usedinterchangeably herein and refers to a nucleotide sequence that iscomplementary to one strand (nucleotide sequence) of a double strand DNAtarget site. The % complementation between the first nucleotide sequencedomain (VT domain) and the target sequence can be at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable target domain can beat least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length. In some embodiments, the variabletargeting domain comprises a contiguous stretch of 12 to 30 nucleotides.

The variable targeting domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence (see forexample modifications described herein), or any combination thereof.

The term “Cas endonuclease recognition domain” or “CER domain” of aguide polynucleotide is used interchangeably herein and refers to anucleotide sequence (such as a second nucleotide sequence domain of aguide polynucleotide), that interacts with a Cas endonucleasepolypeptide. The CER domain can be composed of a DNA sequence, a RNAsequence, a modified DNA sequence, a modified RNA sequence (see forexample modifications described herein), or any combination thereof.

The nucleotide sequence linking the crNucleotide and the tracrNucleotideof a single guide polynucleotide can comprise a RNA sequence, a DNAsequence, or a RNA-DNA combination sequence. In one embodiment, thenucleotide sequence linking the crNucleotide and the tracrNucleotide ofa single guide polynucleotide can be at least 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 nucleotides in length. In one embodiment, the nucleotide sequencelinking the crNucleotide and the tracrNucleotide of a single guidepolynucleotide can comprise a tetraloop sequence, such as, but notlimiting to a GAAA tetraloop sequence.

Nucleotide sequence modification of the guide polynucleotide, VT domainand/or CER domain can be selected from, but not limited to, the groupconsisting of a 5′ cap, a 3′ polyadenylated tail, a riboswitch sequence,a stability control sequence, a sequence that forms a dsRNA duplex, amodification or sequence that targets the guide poly nucleotide to asubcellular location, a modification or sequence that provides fortracking, a modification or sequence that provides a binding site forproteins, a Locked Nucleic Acid (LNA), a 5-methyl dC nucleotide, a2,6-Diaminopurine nucleotide, a 2′-Fluoro A nucleotide, a 2′-Fluoro Unucleotide; a 2′-O-Methyl RNA nucleotide, a phosphorothioate bond,linkage to a cholesterol molecule, linkage to a polyethylene glycolmolecule, linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage,or any combination thereof. These modifications can result in at leastone additional beneficial feature, wherein the additional beneficialfeature is selected from the group of a modified or regulated stability,a subcellular targeting, tracking, a fluorescent label, a binding sitefor a protein or protein complex, modified binding affinity tocomplementary target sequence, modified resistance to cellulardegradation, and increased cellular permeability.

In one embodiment of the disclosure, the composition comprises a guidepolynucleotide comprising: (i) a first nucleotide sequence domain (VTdomain) that is complementary to a nucleotide sequence in a target DNA;and, (ii) a second nucleotide sequence domain (CER domain) thatinteracts with a Cas endonuclease, wherein the first nucleotide sequencedomain and the second nucleotide sequence domain are composed ofdeoxyribonucleic acids (DNA), ribonucleic acids (RNA), or a combinationthereof. The % complementation between the first nucleotide sequencedomain (Variable Targeting domain) and the target sequence can be atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In one embodiment of the disclosure, the first nucleotide sequencedomain (VT domain) and the second nucleotide sequence domain (CERdomain) of the guide polynucleotide are located on a single molecule. Inanother embodiment, the second nucleotide sequence domain (CasEndonuclease Recognition domain) comprises two separate molecules thatare capable of hybridizing along a region of complementarity.

In one embodiment, the composition comprises a guide polynucleotide,wherein the first nucleotide sequence domain (VT domain) is a DNAsequence and the second nucleotide sequence domain (CER domain) isselected from the group consisting of a DNA sequence, a RNA sequence,and a combination thereof.

In one embodiment the guide polynucleotide can be introduce into theplant cell directly using any method known to one skilled in the art,such as for example, but not limited to, particle bombardment or topicalapplications.

When the guide polynucleotide comprises solely of RNA sequences (alsoreferred to as “guide RNA”) it can be introduced indirectly byintroducing a recombinant DNA molecule comprising the correspondingguide DNA sequence operably linked to a plant specific promoter that iscapable of transcribing the guide polynucleotide in said plant cell. Theterm “corresponding guide DNA” refers to a DNA molecule that isidentical to the RNA molecule but has a “T” substituted for each “U” ofthe RNA molecule.

In some embodiments, the guide polynucleotide is introduced via particlebombardment or Agrobacterium transformation of a recombinant DNAconstruct comprising the corresponding guide DNA operably linked to aplant U6 polymerase III promoter.

The terms “target site”, “target sequence”, “target DNA”, “targetlocus”, “genomic target site”, “genomic target sequence”, and “genomictarget locus” are used interchangeably herein and refer to apolynucleotide sequence in the genome (including chloroplastic andmitochondrial DNA) of a cell at which a double-strand break is inducedin the cell genome by a Cas endonuclease. The target site can be anendogenous site in the genome of a cell or organism, or alternatively,the target site can be heterologous to the cell or organism and therebynot be naturally occurring in the genome, or the target site can befound in a heterologous genomic location compared to where it occurs innature. As used herein, terms “endogenous target sequence” and “nativetarget sequence” are used interchangeable herein to refer to a targetsequence that is endogenous or native to the genome of a cell ororganism and is at the endogenous or native position of that targetsequence in the genome of a cell or organism. Cells include, but are notlimited to animal, bacterial, fungal, insect, yeast, and plant cells aswell as plants and seeds produced by the methods described herein.

In one embodiments, the target site, in association with the particulargene editing system that is being used, can be similar to a DNArecognition site or target site that is specifically recognized and/orbound by a double-strand break inducing agent, such as but not limitedto a Zinc Finger endonuclease, a meganuclease, or a TALEN endonuclease.

An “artificial target site” or “artificial target sequence” are usedinterchangeably herein and refer to a target sequence that has beenintroduced into the genome of a cell or organism, such as but notlimiting to a plant or yeast. Such an artificial target sequence can beidentical in sequence to an endogenous or native target sequence in thegenome of a cell but be located in a different position (i.e., anon-endogenous or non-native position) in the genome of a cell ororganism.

An “altered target site”, “altered target sequence”, “modified targetsite”, “modified target sequence” are used interchangeably herein andrefer to a target sequence as disclosed herein that comprises at leastone alteration when compared to non-altered target sequence. Such“alterations” include, for example: (i) replacement of at least onenucleotide, (ii) a deletion of at least one nucleotide, (iii) aninsertion of at least one nucleotide, or (iv) any combination of(i)-(iii).

Polynucleotide constructs that provide a guide RNA which targets anendogenous EPSPS gene of a plant cell are provided herein. Thepolynucleotide construct may further comprise one or more polynucleotidemodification templates to generate a modified endogenous EPSPS gene thatencodes a plant EPSPS polypeptide that comprises G102A and at least oneamino acid mutation selected from the group consisting of: (a) A2R, (b)A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i)K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G,(p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein eachamino acid mutation position corresponds to the amino acid position setforth in SEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodesa polypeptide comprising a sequence that is at least 90% identical toSEQ ID NO:2. The modified endogenous EPSPS gene may encode a plant EPSPSpolypeptide that comprises G102A and at least two, at least three, or atleast four amino acid mutations selected from the group consisting of:(a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f) L98C, (g) K173R, (h)I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S, (m) E391P, (n) E391G,(o) D402G, (p) A416G, (q) V438R, (r) S440R, (s) T441Q, and (t) F442V,wherein each amino acid position corresponds to the amino acid mutationposition set forth in SEQ ID NO:1 and wherein the endogenous plant EPSPSgene encodes a polypeptide comprising a sequence that is at least 90%identical to SEQ ID NO:2. The modified endogenous EPSPS gene may encodea plant EPSPS polypeptide that comprises: (a) A4W, H54M, L98C, G102A,K173R, I208L, K243E, E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V; (b) A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q; or (c) A2R, A4W,K84R, L98C, G102A, I208L, K243E, E391P, and D402G. The modifiedendogenous EPSPS gene may encode a plant EPSPS polypeptide that has theamino acid sequence set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6.

Methods for producing glyphosate tolerant plants are provided herein inwhich a guide RNA, one or more polynucleotide modification templates,and one or more Cas endonucleases are provided to a plant cell. The Casendonuclease(s) introduces a double strand break at an endogenous EPSPSgene in the plant cell, and the polynucleotide modification template(s)is used to generate a modified EPSPS gene that encodes a plant EPSPSpolypeptide that comprises G102A and at least one amino acid mutationselected from the group consisting of: a) A2R, (b) A4W, (c) H54M, (d)A72Q, (e) K84R, (f) L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A,(k) E302S, (l) T361S, (m) E391P, (n) E391G, (o) D402G, (p) A416G, (q)V438R, (r) S440R, (s) T441Q, and (t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2. A plant is obtained from the plant cell, and a glyphosatetolerant progeny plant that is void of the guide RNA and Casendonuclease is generated. The modified endogenous EPSPS gene may encodea plant EPSPS polypeptide that comprises G102A and at least two, atleast three, or at least four amino acid mutations selected from thegroup consisting of: (a) A2R, (b) A4W, (c) H54M, (d) A72Q, (e) K84R, (f)L98C, (g) K173R, (h) I208L, (i) K243E, (j) T279A, (k) E302S, (l) T361S,(m) E391P, (n) E391G, (o) D402G, (p) A416G, (q) V438R, (r) S440R, (s)T441Q, and (t) F442V, wherein each amino acid position corresponds tothe amino acid mutation position set forth in SEQ ID NO:1 and whereinthe endogenous plant EPSPS gene encodes a polypeptide comprising asequence that is at least 90% identical to SEQ ID NO:2. The modifiedendogenous EPSPS gene may encode a plant EPSPS polypeptide thatcomprises: (a) A4W, H54M, L98C, G102A, K173R, I208L, K243E, E302S,T361S, E391P, D402G, A416G, V438R, S440R, T441Q, and F442V; (b) A2R,A4W, A72Q, K84R, L98C, G102A, I208L, T279A, E302S, T361S, E391G, D402G,A416G, V438R, and T441Q; or (c) A2R, A4W, K84R, L98C, G102A, I208L,K243E, E391P, and D402G. The modified endogenous EPSPS gene may encode aplant EPSPS polypeptide that has the amino acid sequence set forth inSEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

Polynucleotide modification templates that are used for endogenousmodification of a gene through CRISPR/Cas9 gene editing are alsoprovided herein. The polynucleotide modification templates may comprisea partial EPSP synthase (EPSPS) sequence and may further comprise one ormore nucleotide mutations that correspond to G102A and to at least oneor more amino acid mutations selected from the group consisting of: a)A2R, b) A4W, c) H54M, d) A72Q, e) K84R, f) L98C, g) K173R, h) I208L, i)K243E, j) T279A, k) E302S, l) T361S, m) E391P, n) E391G, o) D402G, p)A416G, q) V438R, r) S440R, s) T441Q, and t) F442V, wherein each aminoacid mutation position corresponds to the amino acid position set forthin SEQ ID NO: 1, are also provided.

B. Methods for Increasing Expression and/or Activity Level of at LeastOne EPSPS Sequence or an Active Variant or Fragment Thereof in a HostCell of Interest, a Plant or Plant Part

Various methods are provided for the expression of an EPSPS sequence oractive variant or fragment thereof in a host cell of interest. Forexample, the host cell of interest is transformed with the EPSPSsequence and the cells are cultured under conditions which allow for theexpression of the EPSPS sequence. In some embodiments, the cells areharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, or other methods,which are well known to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, 3^(rd) Ed., Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, 4^(th) Ed. W.H. Freeman and Company; andRicciardelli, et al., (1989) In vitro Cell Dev. Biol. 25:1016-1024. Forplant cell culture and regeneration see, Payne et al. (1992) Plant Celland Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York,N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and OrganCulture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin, Heidelberg, New York); Jones, ed. (1984) Plant Gene Transferand Expression Protocols, Humana Press, Totowa, N.J.; and PlantMolecular Biology (1993) R. R. D. Croy, ed. Bios Scientific Publishers,Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are setforth in Atlas and Parks (eds.) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, Fla. Additional information for cellculture is found in available commercial literature such as the LifeScience Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc.(St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., The Plant Culture Catalogueand supplement (1997) also from Sigma-Aldrich, Inc. (St Louis, Mo.)(“Sigma-PCCS”).

A method for increasing the activity of an EPSPS polypeptide disclosedherein or an active variant or fragment thereof in a plant, plant cell,plant part, explant, and/or seed is provided. In further embodiments,the activity of the EPSPS polypeptide is increased in a plant or plantpart by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 500%, 1000%, 5000%, or 10,000% relative to an appropriate controlplant, plant part, or cell. In still other embodiments, the activitylevel of the EPSPS polypeptide in the plant or plant part is increasedby 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 fold or more relative to anappropriate control plant, plant part, or cell. Such an increase in theactivity of the EPSPS polypeptide in the cell can be achieved in avariety of ways including, for example, by the expression of multiplecopies of one or more EPSPS polypeptide, by employing a promoter todrive higher levels of expression of the sequence, or by employing aEPSPS sequence having an increased level of activity.

In specific embodiments, the polypeptide or the EPSPS polynucleotide oractive variant or fragment thereof is introduced into the plant, plantcell, explant or plant part. Subsequently, a plant cell having anintroduced sequence disclosed herein is selected using methods known tothose of skill in the art such as, but not limited to, Southern blotanalysis, DNA sequencing, PCR analysis, or phenotypic analysis. A plantor plant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate thetemporal or spatial expression of polypeptides disclosed herein in theplant. Plant forming conditions are well known in the art and discussedbriefly elsewhere herein.

In one embodiment, a method of producing a glyphosate tolerant plantcell is provided and comprises transforming a plant cell with thepolynucleotide encoding an EPSPS polypeptide or active variant orfragment thereof. In specific embodiments, the method further comprisesselecting a plant cell which is resistant or tolerant to a glyphosate bygrowing the plant cells in a sufficient concentration of glyphosate,such that the herbicide kills the plant cells which do not comprise theEPSPS polypeptide of interest.

C. Method of Producing Crops and Controlling Weeds

Methods for controlling weeds in an area of cultivation, preventing thedevelopment or the appearance of herbicide resistant weeds in an area ofcultivation, producing a crop, and increasing crop safety are provided.The term “controlling,” and derivations thereof, for example, as in“controlling weeds” refers to one or more of inhibiting the growth,germination, reproduction, and/or proliferation of; and/or killing,removing, destroying, or otherwise diminishing the occurrence and/oractivity of a weed.

As used herein, an “area of cultivation” comprises any region in whichone desires to grow a plant. Such areas of cultivations include, but arenot limited to, a field in which a plant is cultivated (such as a cropfield, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc.), a greenhouse, a growth chamber,etc.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 10%, 5%, or 1% of the crop plants are significantly damaged orkilled.

Methods provided comprise planting the area of cultivation with a planthaving a EPSPS sequence or active variant or fragment thereof disclosedherein or transgenic seed derived therefrom, and in specificembodiments, applying to the crop, seed, weed or area of cultivationthereof an effective amount of a herbicide of interest. It is recognizedthat the herbicide can be applied before or after the crop is planted inthe area of cultivation. Such herbicide applications can include anapplication of glyphosate.

Accordingly, the term “glyphosate” should be considered to include anyherbicidally effective form of N-phosphonomethylglycine (including anysalt thereof) and other forms which result in the production of theglyphosate anion in planta.

In specific methods, glyphosate is applied to the plants having theEPSPS sequence or active variant or fragment thereof or their area ofcultivation. In specific embodiments, the glyphosate is in the form of asalt, such as, ammonium, isopropylammonium, potassium, sodium (includingsesquisodium) or trimesium (alternatively named sulfosate). In stillfurther embodiments, a mixture of a synergistically effective amount ofa combination of glyphosate and an ALS inhibitor (such as asulfonylurea) is applied to the plants or their area of cultivation.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. In some embodiments, the effective amount ofglyphosate applied is about 50 gram acid equivalent/acre to about 2000gram acid equivalent/acre. It is important to note that it is notnecessary for the crop to be totally insensitive to the herbicide, solong as the benefit derived from the inhibition of weeds outweighs anynegative impact of the glyphosate or glyphosate analog on the crop orcrop plant.

“Weed” as used herein refers to a plant which is not desirable in aparticular area. Conversely, a “crop plant” as used herein refers to aplant which is desired in a particular area, such as, for example, amaize or soy plant. Thus, in some embodiments, a weed is a non-cropplant or a non-crop species, while in some embodiments, a weed is a cropspecies which is sought to be eliminated from a particular area, suchas, for example, an inferior and/or non-transgenic soy plant in a fieldplanted with a plant having the EPSPS sequence disclosed herein or anactive variant or fragment thereof.

Accordingly, the current disclosure provides methods for selectivelycontrolling weeds in a field containing a crop that involve planting thefield with crop seeds or plants which are glyphosate-tolerant as aresult of being transformed with a gene encoding a EPSPS disclosedherein or an active variant or fragment thereof, and applying to thecrop and weeds in the field a sufficient amount of glyphosate to controlthe weeds without significantly affecting the crop.

Further provided are methods for controlling weeds in a field andpreventing the emergence of glyphosate resistant weeds in a fieldcontaining a crop which involve planting the field with crop seeds orplants that are glyphosate tolerant as a result of being transformedwith a gene encoding EPSPS and a gene encoding a polypeptide impartingglyphosate tolerance by another mechanism, such as, a glyphosatetolerant glyphosate-N-acetyltransferase and/or a glyphosate-tolerantglyphosate oxido-reductase and applying to the crop and the weeds in thefield a sufficient amount of glyphosate to control the weeds withoutsignificantly affecting the crop. Various plants that can be used inthis method are discussed in detail elsewhere herein.

In further embodiments, the current disclosure provides methods forcontrolling weeds in a field and preventing the emergence of herbicideresistant weeds in a field containing a crop which involve planting thefield with crop seeds or plants that are glyphosate tolerant as a resultof being transformed with a gene encoding EPSPS, a gene encoding apolypeptide imparting glyphosate tolerance by another mechanism, suchas, a glyphosate tolerant glyphosate-N-acetyltransferase and/or aglyphosate oxido-reductase and a gene encoding a polypeptide impartingtolerance to an additional herbicide, such as, a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonylurea-tolerant acetolactatesynthase, a sulfonylurea-tolerant acetohydroxy acid synthase, asulfonamide-tolerant acetolactate synthase, a sulfonamide-tolerantacetohydroxy acid synthase, an imidazolinone-tolerant acetolactatesynthase, an imidazolinone-tolerant acetohydroxy acid synthase, aphosphinothricin acetyl transferase and a mutated protoporphyrinogenoxidase and applying to the crop and the weeds in the field a sufficientamount of glyphosate and an additional herbicide, such as, ahydroxyphenylpyruvatedioxygenase inhibitor, sulfonamide, imidazolinone,bialaphos, phosphinothricin, azafenidin, butafenacil, sulfosate,glufosinate, and a protox inhibitor to control the weeds withoutsignificantly affecting the crop. Various plants and seeds that can beused in this method are discussed in detail elsewhere herein.

Further provided are methods for controlling weeds in a field andpreventing the emergence of herbicide resistant weeds in a fieldcontaining a crop which involve planting the field with crop seeds orplants that are glyphosate tolerant as a result of being transformedwith a gene encoding an EPSPS and a gene encoding a polypeptideimparting tolerance to an additional herbicide, such as, a mutatedhydroxyphenylpyruvatedioxygenase, a sulfonamide-tolerant acetolactatesynthase, a sulfonamide-tolerant acetohydroxy acid synthase, animidazolinone-tolerant acetolactate synthase, an imidazolinone-tolerantacetohydroxy acid synthase, a phosphinothricin acetyl transferase and amutated protoporphyrinogen oxidase and applying to the crop and theweeds in the field a sufficient amount of glyphosate and an additionalherbicide, such as, a hydroxyphenylpyruvatedioxygenase inhibitor,sulfonamide, imidazolinone, bialaphos, phosphinothricin, azafenidin,butafenacil, sulfosate, glufosinate, and a protox inhibitor to controlthe weeds without significantly affecting the crop. Various plants andseeds that can be used in this method are discussed in detail elsewhereherein.

Further provided is a method for producing a crop by growing a cropplant that is tolerant to glyphosate as a result of being transformedwith a EPSPS polynucleotide or active variant or fragment thereofdisclosed herein or as a result of the endogenous plant EPSPS gene beingmodified, under conditions such that the crop plant produces a crop, andharvesting the crop. Preferably, the glyphosate is applied to the plant,or in the vicinity of the plant, at a concentration effective to controlweeds without preventing the transgenic crop plant from growing andproducing the crop. The application of the glyphosate can be beforeplanting, or at any time after planting up to and including the time ofharvest. Glyphosate can be applied once or multiple times. The timing ofglyphosate application, amount applied, mode of application, and otherparameters will vary based upon the specific nature of the crop plantand the growing environment, and can be readily determined by one ofskill in the art. A crop produced by this method is also provided.

Further provided are methods for the propagation of a plant containingan EPSPS polypeptide or active variant or fragment thereof. The plantcan be, for example, a monocot or a dicot. In one aspect, propagationentails crossing a plant containing an EPSPS polynucleotide transgenewith a second plant, such that at least some progeny of the crossdisplay glyphosate tolerance.

The methods herein further allow for the development of herbicideapplications to be used with the plants having the EPSPS sequence oractive variants or fragments thereof. In such methods, the environmentalconditions in an area of cultivation are evaluated. Environmentalconditions that can be evaluated include, but are not limited to, groundand surface water pollution concerns, intended use of the crop, croptolerance, soil residuals, weeds present in area of cultivation, soiltexture, pH of soil, amount of organic matter in soil, applicationequipment, and tillage practices. Upon the evaluation of theenvironmental conditions, an effective amount of a combination ofherbicides can be applied to the crop, crop part, and seed of the cropor area of cultivation.

Any herbicide or combination of herbicides can be applied to the planthaving the EPSPS sequence or active variant or fragment thereofdisclosed herein or transgenic seed derived there from, crop part, orthe area of cultivation containing the crop plant. By “treated with acombination of” or “applying a combination of” herbicides to a crop,area of cultivation or field” it is intended that a particular field,crop or weed is treated with each of the herbicides and/or chemicalsindicated to be part of the combination so that a desired effect isachieved, i.e., so that weeds are selectively controlled while the cropis not significantly damaged. The application of each herbicide and/orchemical may be simultaneous or the applications may be at differenttimes (sequential), so long as the desired effect is achieved.Furthermore, the application can occur prior to the planting of thecrop.

Classifications of herbicides (i.e., the grouping of herbicides intoclasses and subclasses) are well-known in the art and includeclassifications by HRAC (Herbicide Resistance Action Committee) and WSSA(the Weed Science Society of America) (see also, Retzinger andMallory-Smith (1997) Weed Technology 11: 384-393).

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application), or by how they are taken up byor affect the plant or by their structure. “Mode of action” generallyrefers to the metabolic or physiological process within the plant thatthe herbicide inhibits or otherwise impairs, whereas “site of action”generally refers to the physical location or biochemical site within theplant where the herbicide acts or directly interacts. Herbicides can beclassified in various ways, including by mode of action and/or site ofaction.

Often, an herbicide-tolerance gene that confers tolerance to aparticular herbicide or other chemical on a plant expressing it willalso confer tolerance to other herbicides or chemicals in the same classor subclass. Thus, in some embodiments, a transgenic plant is tolerantto more than one herbicide or chemical in the same class or subclass,such as, for example, an HPPD inhibitor, glyphosate, an ALS chemistry,an inhibitor of PPO, a sulfonylurea, and/or a synthetic auxin.

Typically, the plants of the present disclosure can tolerate treatmentwith different types of herbicides (i.e., herbicides having differentmodes of action and/or different sites of action) thereby permittingimproved weed management strategies that are recommended in order toreduce the incidence and prevalence of herbicide-tolerant weeds.

In some embodiments, a plant of the disclosure is not significantlydamaged by treatment with a glyphosate herbicide applied to that plantat a dose equivalent to a rate of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 150, 170, 200, 300, 400, 500, 600,700, 800, 800, 1000, 2000, 3000, 4000, 5000, 5400 or more grams orounces (1 ounce=29.57 ml) of active ingredient or commercial product orherbicide formulation per acre or per hectare, whereas an appropriatecontrol plant is significantly damaged by the same glyphosate treatment.

Additional ranges of the effective amounts of herbicides can be found,for example, in various publications from University Extension services.See, for example, Bernards, et al., (2006) Guide for Weed Management inNebraska (www.ianrpubs.url.edu/sendlt/ec130); Regher, et al., (2005)Chemical Weed Control for Fields Crops, Pastures, Rangeland, andNoncropland, Kansas State University Agricultural Extension Station andCorporate Extension Service; Zollinger, et al., (2006) North Dakota WeedControl Guide, North Dakota Extension Service and the Iowa StateUniversity Extension at www.weeds.iastate.edu, each of which is hereinincorporated by reference.

In some embodiments of the disclosure, glyphosate is applied to an areaof cultivation and/or to at least one plant in an area of cultivation atrates between 8 and 32 ounces of acid equivalent per acre, or at ratesbetween 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 ounces of acidequivalent per acre at the lower end of the range of application andbetween 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32 ounces of acidequivalent per acre at the higher end of the range of application (1ounce=29.57 ml). In other embodiments, glyphosate is applied at least at1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater ounce of activeingredient per hectare (1 ounce=29.57 ml). In some embodiments of thedisclosure, a sulfonylurea herbicide is applied to a field and/or to atleast one plant in a field at rates between 0.04 and 1.0 ounces ofactive ingredient per acre, or at rates between 0.1, 0.2, 0.4, 0.6 and0.8 ounces of active ingredient per acre at the lower end of the rangeof application and between 0.2, 0.4, 0.6, 0.8 and 1.0 ounces of activeingredient per acre at the higher end of the range of application. (1ounce=29.57 ml). In some embodiments, as described herein, glyphosatetreatment can be made at a stage starting from pre-emergence to earlyreproductive stages of the crop plant for weed control.

III. A Rapid Assay for Catalytic Efficiency of a Plurality of EnzymeVariants

One of the commercial applications of directed evolution is todesensitize an enzyme to inhibition by, for example, a herbicide. kcat,1/KM, and KI are three dimensions that when multiplied are a measure ofan enzyme's intrinsic capacity for catalysis in the presence of aninhibitor. The ideal values for the individual dimensions depend onsubstrate and inhibitor concentrations under the conditions of theapplication. When attempting to optimize those values by directedevolution, (kcat/KM)*KI can be an informative parameter for evaluatinglibraries of variants. However, evaluating (kcat/KM)*KI for hundreds ofvariants by substrate saturation analysis may not provide adequatethroughput. A manipulation of the Michaelis-Menten equation that enablesisolation of (kcat/KM)*KI on one side of the equation is presentedherein. If substrate and enzyme concentrations are identical butvelocity is measured at two different inhibitor concentrations (one ofwhich can be 0), the data are sufficient to calculate (kcat/KM)*KI withjust two rate measurements. The procedure has been validated bycorrelating values obtained with the rapid method with those obtained bysubstrate saturation kinetics.

The method includes (a) providing a plurality of enzyme variants; (b)providing the inhibitor; (c) providing the substrate; (d) performing areaction involving the plurality of enzyme variants and the substrate,at no more than two different inhibitor concentrations; (e) measuringreaction rate at no more than two different inhibitor concentrations;and (f) calculating (kcat/KM)*KI of the plurality of enzyme variants. Insome embodiments, one of the inhibitor concentrations is zero. In otherembodiments, the substrate is at a concentration that is substantiallysimilar to Michaelis-Menten constant (KM) of a parental enzyme for theenzyme variant. In still other embodiments, the enzyme is at asufficient concentration to result in a substantially linear reactionrate at the two different inhibitor concentrations. In still otherembodiments, one of the inhibitor concentrations is sufficient to resultin at least about 50% inhibition. In still other embodiments, the assayis performed in a high-throughput system. In still other embodiments,the catalytic capacity in the presence of the inhibitor is estimated byobtaining a numerical value for (kcat/KM)*KI, wherein kcat is maximumenzyme turnover rate, KM is Michaelis-Menten constant and KI isinhibitor dissociation constant. In some embodiments, the substrate isPEP; the inhibitor is glyphosate; and the plurality of enzyme variantsare EPSPS enzyme variants. In still other embodiments, the enzyme andthe substrate concentrations are the same, at the two inhibitorconcentrations.

EXAMPLES

In the following Examples, unless otherwise stated, in which parts andpercentages are by weight and degrees are Celsius. It should beunderstood that these Examples, while indicating embodiments of theinvention, are given by way of illustration only. From the abovediscussion and these Examples, one skilled in the art, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions. Such modifications are also intended to fall within thescope of the appended embodiments.

Example 1 Expression, Purification, and Assessment of Plant EPSPSynthases

The amino acid sequence of mature Zea mays EPSP synthase (EPSPS) wasobtained from GenBank entry CAA44974.1 (NCBI GI No. 1524383; presentedherein as SEQ ID NO:2). A nucleotide sequence was created to add anN-terminal methionine and to optimize codon usage for expression in E.coli. The synthetic gene was supplied by a commercial vendor. The genewas cloned into an expression vector that provides a T7 promoter drivingexpression of the protein. The vector was modified to change a6×N-terminal histidine tag to a 10×tag. The resulting coding region ofthe vector yields an expressed protein with the amino acid sequenceshown in Table 1 (represented by SEQ ID NO:1). The coding region ispreceded by an N-terminal extension represented by SEQ ID NO:3.

TABLE 1 Amino acid sequence of the variant termed “native maize EPSPS”.This sequence is the reference for all position numbers provided hereinas it relates to maize EPSPS mutations disclosed herein. 1 2 3 4 5 6 7 89 10 11 12 13 14 15 M A G A E E I V L Q P I K E I 16 17 18 19 20 21 2223 24 25 26 27 28 29 30 S G T V K L P G S K S L S N R 31 32 33 34 35 3637 38 39 40 41 42 43 44 45 I L L L A A L S E G T T V V D 46 47 48 49 5051 52 53 54 55 56 57 58 59 60 N L L N S E D V H Y M L G A L 61 62 63 6465 66 67 68 69 70 71 72 73 74 75 R T L G L S V E A D K A A K R 76 77 7879 80 81 82 83 84 85 86 87 88 89 90 A V V V G C G G K F P V E D A 91 9293 94 95 96 97 98 99 100 101 102 103 104 105 K E E V Q L F L G N A G T AM 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 R P L T AA V T A A G G N A T 121 122 123 124 125 126 127 128 129 130 131 132 133134 135 Y V L D G V P R M R E R P I G 136 137 138 139 140 141 142 143144 145 146 147 148 149 150 D L V V G L K Q L G A D V D C 151 152 153154 155 156 157 158 159 160 161 162 163 164 165 F L G T D C P P V R V NG I G 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 G L PG G K V K L S G S I S S 181 182 183 184 185 186 187 188 189 190 191 192193 194 195 Q Y L S A L L M A A P L A L G 196 197 198 199 200 201 202203 204 205 206 207 208 209 210 D V E I E I I D K L I S I P Y 211 212213 214 215 216 217 218 219 220 221 222 223 224 225 V E M T L R L M E RF G V K A 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 EH S D S W D R F Y I K G G Q 241 242 243 244 245 246 247 248 249 250 251252 253 254 255 K Y K S P K N A Y V E G D A S 256 257 258 259 260 261262 263 264 265 266 267 268 269 270 S A S Y F L A G A A I T G G T 271272 273 274 275 276 277 278 279 280 281 282 283 284 285 V T V E G C G TT S L Q G D V 286 287 288 289 290 291 292 293 294 295 296 297 298 299300 K F A E V L E M M G A K V T W 301 302 303 304 305 306 307 308 309310 311 312 313 314 315 T E T S V T V T G P P R E P F 316 317 318 319320 321 322 323 324 325 326 327 328 329 330 G R K H L K A I D V N M N KM 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 P D V A MT L A V V A L F A D 346 347 348 349 350 351 352 353 354 355 356 357 358359 360 G P T A I R D V A S W R V K E 361 362 363 364 365 366 367 368369 370 371 372 373 374 375 T E R M V A I R T E L T K L G 376 377 378379 380 381 382 383 384 385 386 387 388 389 390 A S V E E G P D Y C I IT P P 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 E K LN V T A I D T Y D D H R 406 407 408 409 410 411 412 413 414 415 416 417418 419 420 M A M A F S L A A C A E V P V 421 422 423 424 425 426 427428 429 430 431 432 433 434 435 T I R D P G C T R K T F P D Y 436 437438 439 440 441 442 443 444 445 F D V L S T F V K N

AroA Knock-Out Strain

The E. coli gene coding for EPSPS (AroA) was functionally deleted by P1phage viral transduction. The donor strain was JW0891 (CGSC), in whichthe AroA gene is disrupted with a Kanamycin resistant gene. Therecipient strain was BI21DE3-Tuner (Novagen). Virus particles werepropagated in untransformed Top10 cells. A stock of 10⁹ plaque formingunits was diluted 1:10 and 1:100. From each dilution, 10 uL of phage wasadded to 0.3 ml of JW0891 donor cells at a density of 0.4 OD. After 30min at 37° C., the mixture of phage and cells was plated on top agar(0.6%) and grown overnight at 37° C. Five ml of liquid LB mediumcontaining 10 mM CaCl₂ was added to the top agar to harvest the plaques.The collected liquid was combined with 1 ml of chloroform and mixedthoroughly. The mixture was then spun and the supernatant was stored at4° C. Transduction mixtures contained 0.3 ml of BL21(DE3) Tuner cells inLB at a density of 0.5 OD, 10 mM CaCl₂ and 10 ul of virus particlesharvested from the donor cells, at 10-, 100- and 1000-fold dilution.Transduction was allowed to proceed at 37° C. for 1 hour. 120 ul of LBcontaining 100 mM sodium citrate was added to each cell and phagemixture and the mixtures were shaken for 1 hour at 37° C. Cells wereplated on selective medium containing 40 mg/L Kanamycin and sodiumcitrate. Transduction of the disrupted AroA gene into Tuner wasconfirmed by sequencing the region of the AroA gene. The Tuner knockoutstrain was made electro-competent by washing and resuspending in 10%glycerol.

EPSPS Production and Purification

Whether protein production was done in BL21(DE3) or with the Tuner AroAknockout, the vector was electroporated into cells and transformantswere selected for growth on LB agar containing 100 ug carbennicillin/ml.Cells were grown in Magic Medium, in which induction occurs when themedium becomes depleted of glucose. After 4 hours of growth at 37° C.,cells were transferred to 30° C. and grown another 16 hrs. Pelletedcells were lysed with BPER (Pierce) protein extraction reagentcontaining 0.2 mg/ml lysozyme, 1 mM dithiothreitol, protease inhibitorcocktail (Sigma, bacterial cocktail) and endonuclease. Insolublecellular debris was removed by centrifugation. EPSPS protein waspurified from the soluble protein solution by affinity chromatography onthe nickel form of nitrilotriacetic acid (Ni-NTA) resin (Qiagen).Protein concentration was measured by absorbance at 280 nm using anextinction coefficient of 0.676 OD/mg/ml, provided by Vector NTI.

EPSPS Assay

Shikimate-3-phosphate (S3P) was prepared from cultures of Klebsiellapneumonia aroA-(ATCC 25597). Cells from a 500 ml culture grown in 2×YTwere used to inoculate 6 L of minimal medium augmented with 55 uMtyrosine, 60 uM phenylalanine, 25 uM tryptophan, 0.1 uM 4-aminobenzoateand 0.1 uM 4-hydroxybenzoate (Weiss et al., 1953. J Amer Chem Soc75:5572-5576). Accumulation of S3P was monitored by anion exchange HPLC.After about 4 days shaking at 37 C, the concentration reached ˜1 mM. S3Pwas purified from the culture supernatant by anion exchangechromatography in ammonium bicarbonate at pH 7.3, with gradient elutionup to 0.7 M. S3P was cleanly separated from phosphate, which elutedearlier.

EPSPS activity was determined by quantifying the phosphate generatedfrom the EPSPS reaction. Release of inorganic phosphate was coupled toreaction with 2-amino-6-mercapto-7-methylpurine ribonucleoside,catalyzed by purine-nucleoside phosphorylase (M R Webb, Proc. Natl.Acad. Sci. 89:4884-4887, 1992). The absorbance change that occurs wasmonitored at 360 nm, where the extinction is 11,200 M⁻¹ cm⁻¹, with aSpectramax plate reader (Molecular Devices). To determine kineticparameters, the varied substrate was present at seven concentrations(the eighth being the blank, containing no substrate) ranging from 4 to400 uM and the unvaried substrate present at saturation. Six microlitersof 50-fold concentrated stock solutions of the varied substrate wereplaced in the wells of the 96-well assay plate and reactions werestarted with the addition of a mixture containing 25 mM Hepes, pH 7, 100mM KCl, 0.3 mM 2-amino-6-mercapto-7-methylpurine ribonucleosde, 1 uM (1unit/ml) purine-nucleoside phosphorylase (Sigma N8264) and 200 uM of thenon-varied substrate. Reactions were monitored with a Spectramax platereader. The Michaelis-Menten kinetics protocol of the Spectramaxsoftware was customized for the substrate concentrations used. Thesoftware returns values of K_(M) and V_(max) using the Lineweaver-Burketransformation of the Michaelis-Menten equation. To determine k_(cat),V_(max) (uM/min) was divided by the enzyme concentration (uM). Todetermine K_(I), substrate saturation was repeated at a higher range ofPEP concentrations in the presence of a concentration of glyphosate thatyielded approximately 2-fold elevation in apparent K_(M) for PEP. K_(I)was then calculated from the following form of the Michaelis-Mentenequation for competitive inhibition:

K _(I) =K _(M)[I]/(K _(M app) −K _(M))

K_(M) approximates the dissociation constant of the enzyme-substratecomplex while k_(cat) is the rate of conversion of substrate to productwhen the substrate concentration is saturating. k_(cat)/K_(M) is awidely accepted parameter for catalytic efficiency when substrateconcentration is low (˜K_(M)). K_(I) is the dissociation constant of theenzyme-inhibitor complex, with a higher value indicating that theequilibrium lies more toward free enzyme and higher insensitivity. Theparameter k_(cat)/K_(M)×K_(I) is used to quantify both catalyticefficiency and insensitivity and thus the overall fitness of the enzyme.

Example 2 Directed Evolution of Maize EPSPS Saturation Mutagenesis

The mature form of native maize EPSPS was subjected to saturationmutagenesis to discover novel mutations that reduce sensitivity toglyphosate. Libraries of substitutions for each position in the EPSPSpolypeptide chain were created using NNK (where N represents a 25% mixeach of adenine, thymine, guanine, and cytosine nucleotides; and Krepresents a 50% mix each of thymine and guanine nucleotides) as thedegenerate codon for the position to be mutagenized. PCR reactionmixtures contained a mutagenic forward primer (NNK codon flanked by 28nucleotides matching with template at each side of the NNK) and areverse primer that was the complement of the sequence preceding theforward primer, 28 nucleotides in length. To make circular doublestranded DNA plasmids from the blunt ended PCR products, the productswere digested with T4 polynucleotide kinase, T4 DNA ligase, and Dpnl (todisrupt the parental DNA template). After desalting by ultrafiltration,the ligation products were ready for transformation and downstreamapplications.

Screen for Beneficial Mutations

The requirement for flux through the EPSPS reaction for growth onminimal medium is a powerful selection for a functional EPSPS expressedfrom a plasmid, provided that the native AroA gene is knocked out. Thus,the Tuner knock out strain was used in the early phase of optimizationwhere insensitivity to glyphosate would be in a range similar to that ofthe endogenous EPSPS. Single mutations in native EPSPS were not expectedto confer significant insensitivity to glyphosate, so the glyphosateconcentration in the selection medium was a relatively low concentrationof 10 mM. Colonies were isolated and heterologously expressed EPSPS wasproduced and purified as above. Purified variants were then assayed bymeasuring reaction rates in the presence of 50 uM phosphoenolpyruvate(PEP) and shikimate-3-phosphate (S3P), with or without 10 uM glyphosate.Mutations beneficial for activity or insensitivity to glyphosate areshown in Table 2.

TABLE 2 Activity of maize EPSPS variants in the presence of 50 uM PEPand shikimate-3-phosphate, with or without the addition of 10 uMglyphosate. Values represent the rate of phosphate formation (uM permin) per uM EPSPS. The rate in the second column is the rate in thepresence of 10 μM glyphosate, expressed as a % of the rate with noglyphosate. Reaction rate No 10 μM glyph, Variant glyph % no glyph 224R249V 768 3.09 241A 278G 730 3.75 311S 701 4.37 4R 662 7.38 278V 622 3.25208V 313G 607 3.21 202R 586 7.48 4N 571 7.56 194M 508 11.5 78L 506 13.4328C 497 11.1 6S 489 8.29 437R 481 11.3 402G 465 19.1 4L 431 13.2 Maizewild- 424 9.49 type 76T 404 18.8 438R 377 19.1 2P 365 13.3 445G 335 18.5313G 330 9.04 310V 323 7.98 391G 317 10.5 338S 294 15.2 101S 216 29.1302S 189 19.3 107S 179 33.2 156Y 170 28.8 107G 161 45.4 156G 148 30.0246G 130 23.7 107L 120 51.7 107V 51 71.7 107Q 37 95.4

Saturation Mutagenesis at Positions 103 and 107

NNK-enabled saturation mutagenesis was performed at positions 103 and107 in the maize EPSPS sequence, and transformed BL21 DE3 cells werescreened as described above. Representative results are presented inTable 3.

TABLE 3 Kinetic parameters of variants selected after simultaneoussaturation mutagenesis at positions 103 and 107 103-107 k_(cat) K_(M)k_(cat)/K_(M) K_(I) k_(cat)/K_(M)*K_(I) G-W 379 82.2 4.62 403 1862 L-T184 45.8 4.02 289 1163 L-A 125 50.6 2.47 463 1143 S-N 161 38.1 4.23 2441033 V-G 324 133 2.44 480 1170 Native 1464 15.7 93.8 0.13 11.8The EPSPS-TGPW protein version is about 3,000 fold less sensitive toglyphosate. However, its catalytic efficiency (k_(cat)/K_(M)) is only 5%of that of native maize EPSPS due to its 4-fold lower k_(cat) and 5-foldhigher K_(M) for PEP.

Combinatorial Shuffling of Native Maize EPSPS

The preceding investigation revealed no novel single mutations thatconfer significant insensitivity to glyphosate, nor any novelcombinations of amino acids at positions 103 and 107 that conferinsensitivity while preserving catalytic efficiency. However, somecombination of the substitutions identified may yield a variant with thedesired properties. Therefore a combinatorial library was designed andsynthesized. The complete list of variable amino acid positions thatwere randomly combined in the library is shown in Table 4.

TABLE 4 Diversity used to construct combinatorial library WT-FS. Shownare the position numbers and the substitutions in the native maize EPSPS(SEQ ID NO: 1). 2P 4LNR 6S 76T 78L 101s 102A 103IALGV 107GLQSWA 156GY194M 202R 208V 224R 241A 246G 249V 278VG 302S 310V 311S 313G 328C 338S391G 402G 437R 438R 445GThe diversity used in combinatorial library WT-FS is the same diversityshown in Tables 2 and 3 with the addition of G102A, 103A and 103I. 102Awas added because of its known effect in desensitizing EPSPS toglyphosate (Sost and Amrhein. 1990. Arch Biochem Biophy 282:433-436;Eschenburg et al. 2002. Planta 216:129-35). The library was synthesizedby fully synthetic shuffling (Ness, J. E. et al. 2002. NatureBiotechnology 20:1251-1255). The theoretical number of unique membersthe library could comprise assuming 1 to 10 mutations per gene is5.6×10⁸.

The vector DNA of the library was transformed into the BL21(DE3) TunerAroA knockout strain and the cells were plated onto M9 medium containing50 mM glyphosate. A small aliquot of the transformed cells was plated onLB, by which it was determined that 1.1×10⁸ colony forming units, or 20%of the theoretical library size, were plated and screened. 115 colonieswere picked and subjected to a second tier of screening in which EPSPSproteins were purified as described in Example 1 and activity measuredunder three conditions: 0.2 mM PEP and S3P, 0.05 mM PEP and S3P, and0.05 mM PEP and S3P plus 10 uM glyphosate. Hits were selected byconsidering the reaction rate at high substrate concentrations(reflecting k_(cat)), the ratio of activity at low to high substrateconcentrations (reflecting K_(M)), and the ratio of activity with towithout glyphosate (reflecting K_(I)). The selected variants weresubjected to substrate saturation kinetic analysis as described inExample 1. Kinetic parameters for selected variants are shown in Table5.

TABLE 5 Kinetic parameters for variants selected from the WT-FScombinatorial library (see text for description) Mutations on wild-k_(cat) K_(M) k_(cat/)K_(M) K_(I) Variant type min⁻¹ uM min⁻¹uM⁻¹ uMK_(cat/)K_(M)*K_(I) WT-FS-B 103I 107S 278G 97.2 27.7 3.57 552 1971 338SWT-FS-D 101S 107L 302S 1853 20.5 92.3 3.81 346 338S WT-FS-E 102A 302S391G 398 165 2.41 1146 2763 438R WT-FS-E2 102A 302S 391G 596 87.7 6.80663 4512 438R I208L G102A 102A 695 310 2.2 2290 5137 P107L 107L 145267.1 22.1 3.6 79 Native 1464 15.7 93.8 0.13 11.8

Variant B contains the T103I and P107S mutations present in the GA21maize transformation event (e.g. U.S. Pat. Nos. 6,566,587 and6,040,497). Kinetic analysis indicates that the TIPS mutations confer ahigh level of insensitivity to glyphosate while retaining near nativeaffinity for PEP but with only ˜5% of the native kcat (Funke et al.2009. J Biological Chemistry 284:9854-9860; Yu et al. 2015. PlantPhysiology. February 2015 pp. 00146.2015). Step-wise acquisition of bothT103I and P107S mutations was documented in a population of Eleusineindica (Yu et al. 2015 supra). However, out of a population of 193individual plants, only 1.6% were homozygous for TIPS, indicating thatthe normal catalytic capacity contributed from the P107S allele was moreimportant for fitness than having the second allele contribute a highlyinsensitive, but catalytically deficient enzyme.

Variant E has alanine substituted for glycine at position 102. Alanineis present at the homologous position in the Type II EPSPS fromAgrobacterium sp. Strain CP4, an enzyme with a high degree ofinsensitivity to glyphosate combined with a low K_(M) for PEP of 12 uM(U.S. Pat. No. 5,633,435). Because PEP is shorter than glyphosate, it ishypothesized that the alanine methyl group in CP4 EPSPS is suitablypositioned to interfere with binding of glyphosate but not PEP. There isonly 24-26% homology between the CP4 enzyme and E. coli or maize EPSPS(U.S. Pat. No. 5,633,435).

The three additional mutations in variant WT-FS-E compared to G102Aalone (Table 5) already confer a 2-fold improvement in K_(M) for PEP.From those observations, it was reasoned that further mutagenesis ofvariant FS-WT-E could result in a context for the A102 methyl group suchthat its position could provide favorable kinetic parameters.

Likewise, the additional mutations present in variant WT-FS-D versusP107L alone provided improved kinetic parameters (Table 5). Furtheroptimization of variants WT-FS-B, -D and -E, representing each of thepreviously known mechanisms for rendering an EPSPS that is lesssensitive to inhibition by glyphosate, was attempted. The objectiveswere to increase the k_(cat) of B, increase the K_(I) of D and improvek_(cat)/K_(M) for PEP of E. Each was subjected to saturation mutagenesisas described in Example 1. The neutral or beneficial singlesubstitutions identified for each is shown in Table 6. (The tableincludes the results from saturation mutagenesis of variant 868-H6,discussed below.)

TABLE 6 Amino acid sequence diversity resulting from saturationmutagenesis of native maize EPSPS and variants WT-FS-B, WT-FS-D, WT-FS-Eand 868-H6. Only the positions that vary from native maize EPSPS areincluded. Bold indicates the amino acids that were present in thebackbone sequence prior to saturation mutagenesis. AA in Backbonesequence for mutagenesis native Position Native B D E H6 A 2 P R R A 4LNR PVW W E 6 S A 36 M G E 39 G N 46 E H 54 W R M E L 65 V A 69 V A 72 RVP GQ QE K 74 V A 76 T V V 78 L K 84 R R V 87 T D 89 F K 91 G E 92 G L98 C C A 101 S S G 102 AG A A T 103 IALGV I P 107 GLQSWA S L A 111 V N118 C D 124 N V 126 A P 127 R E 131 L L 137 M Q 143 G L 152 VY G C 156GY I 164 V K 171 G K 173 G G R S 177 M M 188 C A 190 S S L 194 M D 196 EE 202 R I 208 V LASREG L R 216 A K 224 R DN E RQ R 233 M G 239 E M K 241A AV K 243 W K 246 G G N 247 LQ Y 249 V E 274 Q T 279 A A T 278 VG GTV K297 SR E 302 S S S S T 308 A P 310 V P 311 S E 313 G N 328 C R A 338 SAS S A 349 I T 361 S S P 382 E D E 391 G G G D 402 G G A 416 G Y 435 F D437 R V 438 R R R T 441 Q N 445 GThere are several positions (54, 72, 173, and 224) at which saturationmutagenesis yielded neutral or beneficial mutations in multiplebackbones. However, in most cases, the particular amino acidsubstitution was specific for a particular backbone. At only sixpositions (173, 190, 224, 241, 246, and 278) was the identicalsubstitution found in more than two of the five backbones, and in nocase was one found in four backbones. The general case is that there islittle overlap in the neutral or beneficial single mutations among thebackbones in which the diversity is generated, indicating that theimpact on the fitness of the enzyme that accrues from a mutation dependson the sequence context in which the mutation occurs. Given the sequencehomology among various plant EPSP synthases, the corresponding sequencecontext of the specific mutations provided herein for the maize EPSPSsequence, are readily ascertainable in another species such as rice,sorghum, and sunflower, based on the guidance of this disclosure. It issignificant to note that none of the changes shown in Table 6 confersmore than 2-fold improvement in k_(cat)/K_(M)×K_(I), found with theI208L mutation in variant WT-FS-E (Table 5).

From the functional diversity identified for each, combinatoriallibraries were constructed and screened. In the case of WT-FS-E, theI208L mutation was fixed into the backbone of the combinatorial library.Kinetic parameters of selected hits from the three combinatoriallibraries are shown in Table 7.

TABLE 7 Kinetic parameters of selected variants following saturationmutagenesis and combinatorial shuffling of EPSPS variants FS-WT-B, -Dand -E. k_(cat), min⁻¹ K_(M), uM k_(cat)/K_(M) K_(I), uMk_(cat)/K_(M)*K_(I) Native 1464 15.7 93.8 0.13 12 WT-FS-E2 596 87.7 6.80663 4512 771-C2 348 21.0 16.5 350 5800 868-H6 386 24.0 16.1 629 10070123-C1 438 24.2 18.1 559 10100The improvements intended for FS-WT-B and -D, namely, improved k_(cat)and K_(I), respectively, did not ensue from one round of diversitygeneration and combinatorial shuffling and no attempt was made tofurther improve them (data not shown). However, two variants, 771-C2 and868-H6 (herein also referred to as “C2” and “H6”, respectively), weresignificantly improved relative to FS-WT-E-I208L. To fully explore thediversity present in 771-C2 and 868-H6, a library was constructed inwhich the variable positions of both enzymes were toggled with thenative amino acid. This would allow all positions to acquire asubstitution or revert to the native amino acid, thus creating allpossible combinations of the mutation present in the two variants andeliminating non-essential or deleterious mutations. The design of thelibrary is seen in Table 8. Semi-synthetic shuffling was used to togglethe diversity at each position shown among the amino acids present innative maize EPSPS, 771-C2 and 868-H6. The procedure did not result insignificant improvement. However, variant 123-C1 (herein also referredto as “C1) has kinetic parameters that were modestly improved over868-H6, while having only 9 mutations compared with 15 for 868-H6. The123-C1 sequence is shown in Table 8 and its kinetic data in Table 7.

TABLE 8 Diversity associated with descendants of variant WT-FS-E Variant2 4 54 72 84 98 102 173 208 243 native A A H A K L G K I K 771-C2 A W*M+ A K C* A* R+ L* E* 868-H6 R* W* H Q+ R* C* A* K L* K 123-C1 R* W* H AR* C* A* K L* E* Variant 279 302 361 391 402 416 438 440 441 442 nativeT E T E D A V S T F 771-C2 T S* S* P* G* G* R* R+ Q* V+ 868-H6 A+ S* S*G+ G* G R S Q* F 123-C1 T E T P G* A V S T F No fill: native +unique tothe variant *Asterisk: shared by more than one variant

Example 3 Mutations of 868-H6 and 123-C1 are Transferable to EPSPS fromOther Plant Species

An alignment of the amino acid sequences of EPSPS from various plantspecies shows a level of homology ranging from 80% to 99%, suggestingthat the mutations defined in the maize background would have a similareffect in EPSPS from other species. The alignment in Table 9 was used tomap the 868-H6 and 123-C1 mutations onto the sequences shown.

Native EPSPS amino acid sequences of rice (Oryza sativa) (SEQ ID NO:7),sorghum (Sorghum halepense) (SEQ ID NO:8), and sunflower (Helianthusannus) (SEQ ID NO:9) including the chloroplast transit peptide sequenceswere assembled and analyzed for mapping the corresponding amino acidmutations from the maize 868-H6. If complete EPSPS sequences were notavailable, appropriate adjustments were made based on sequencealignments and conserved residue mapping.

There is no complete sequence available for Helianthus annuus. Thesequence shown below is a composite of a partial H. annuus sequence(GE499295) coding for amino acids 61-323 in Table 9 and cDNA data for H.salicifolius (AY545662.1), H. ciliaris (EL428089) and TC22032(unidentified species).

Table 9 represents the mapping of maize 868-H6 EPSPS mutations ontoEPSPS from other crop species. The 15 mutations present in 868H6 areshown in reverse highlight. Zm: Zea mays; Hel an: Helianthus annuus; Orysa: Oryza sativa; Sor ha: Sorghum halepense.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Zm — A G A E E I V LQ P I K E I S G T V K Zm H6, C1 M R* G W* E E I V L Q P I K E I S G T VK Hel an S T A P E E I V L K P I K E I S G T V N Ory sa A A K A E E I VL Q P I R E I S G A V Q Sor ha — A G A E E I V L Q P I K E I S G T V K21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Zm L P G S KS L S N R I L L L A A L S E G Zm H6, C1 L P G S K S L S N R I L L L A AL S E G Hel an L P G S K S L S N R I L L L A A L A E G Ory sa L P G S KS L S N R I L L L S A L S E G Sor ha L P G S K S L S N R I L L L A A L SE G 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Zm T T VV D N L L N S E D V H Y M L G A L Zm H6, C1 T T V V D N L L N S E D V HY M L G A L Hel an T T V V D N L L N S D D V H Y M L G A L Ory sa T T VV D N L L N S E D V H Y M L E A L Sor ha T T V V D N L L N S E D V H Y ML G A L 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 Zm RT L G L S V E A D K A A K R A V V V G Zm H6, C1 R T L G L S V E A D K QA K R A V V V G Hel an R A L G L N V E E N G E I K R A T V E G Ory sa KA L G L S V E A D K V A K R A V V V G Sor ha N T L G L S V E A D K V A KR A V V V G 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 ZmC G G K F P V E — D A K E E V Q L F L G Zm H6, C1 C G G R* F P V E — D AK E E V Q L F C* G Hel an C G G V F P V G K E A K D E I Q L F L G Ory saC G G K F P V E K D A K E E V Q L F L G Sor ha C G G K F P V E — D A K EE V Q L F L G 100 101 102 103 104 105 106 107 108 109 110 111 112 113114 115 116 117 118 119 Zm N A G T A M R P L T A A V T A A G G N A ZmH6, C1 N A R* T A M R P L T A A V T A A G G N A Hel an N A G T A M R P LT A A V T A A G G N S Ory sa N A G T A M R P L T A A V T A A G G N A Sorha N A G T A M R P L T A A V T A A G G N A 120 121 122 123 124 125 126127 128 129 130 131 132 133 134 135 136 137 138 139 Zm T Y V L D G V P RM R E R P I G D L V V Zm H6, C1 T Y V L D G V P R M R E R P I G D L V VHel an S Y I L D G V P R M R E R P I G D L V T Ory sa T Y V L D G V P RM R E R P I G D L V V Sor ha T Y V L D G V P R M R E R P I G D L V V 140141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158159 Zm G L K Q L G A D V D C F L G T D C P P V Zm H6, C1 G L K Q L G A DV D C F L G T D C P P V Hel an G L K Q L G A D V D C F L G T N C P P VOry sa G L K Q L G A D V D C F L G T E C P P V Sor ha G L K Q L G A D VD C F L G T D C P P V 160 161 162 163 164 165 166 167 168 169 170 171172 173 174 175 176 177 178 179 Zm R V N G I G G L P G G K V K L S G S IS Zm H6 R V N G I G G L P G G K V K L S G S I S Hel an R V A A N G G L PG G K V K L S G S I S Ory sa R V K G I G G L P G G K V K L S G S I S Sorha R I N G I G G L P G G K V K L S G S I S 180 181 182 183 184 185 186187 188 189 190 191 192 193 194 195 196 197 198 199 Zm S Q Y L S A L L MA A P L A L G D V E I Zm H6, C1 S Q Y L S A L L M A A P L A L G D V E IHel an S Q Y L T A L L M A A P L A L G D V E I Ory sa S Q Y L S A L L MA A P L A L G D V E I Sor ha S Q Y L S A L L M A A P L A L G D V E I 200201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218219 Zm E I I D K L I S I P Y V E M T L R L M E Zm H6, C1 E I I D K L I SL* P Y V E M T L R L M E Hel an E I I D K L I S V P Y V E M T L K L M EOry sa E I I D K L I S I P Y V E M T L R L M E Sor ha E I I D K L I S IP Y V E M T L R L M E 220 221 222 223 224 225 226 227 228 229 230 231232 233 234 235 236 237 238 239 Zm R F G V K A E H S D S W D R F Y I K GG Zm H6, C1 R F G V K A E H S D S W D R F Y I K G G Hel an R F G V S V EH S D S W D K F Y V R G G Ory sa R F G V K A E H S D S W D R F Y I K G GSor ha R F G V K A E H S D S W D R F Y I K G G 240 241 242 243 244 245246 247 248 249 250 251 252 253 254 255 256 257 258 259 Zm Q K Y K S P KN A Y V E G D A S S A S Y Zm H6, C1 Q K Y E S P K N A Y V E G D A S S AS Y Hel an Q K Y K S P G N A Y V E G D A S S A S Y Ory sa Q K Y K S P GN A Y V E G D A S S A S Y Sor ha Q K Y K S P K N A Y V E G D A S S A S Y260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277278 279 Zm F L A G A A I T G G T V T V E G C G T T Zm H6, C1 F L A G A AI T G G T V T V E G C G T A Hel an F L A G A A I T G G T V T V E G C G TS Ory sa F L A G A A I T G G T V T V Q G C G T T Sor ha F L A G A A I TG G T V T V E G C G T T 280 281 282 283 284 285 286 287 288 289 290 291292 293 294 295 296 297 298 299 Zm S L Q G D V K F A E V L E M M G A K VT Zm H6, C1 S L Q G D V K F A E V L E M M G A K V T Hel an S L Q G D V KF A E V L G Q M G A E V T Ory sa S L Q G D V K F A E V L E M M G A K V TSor ha S L Q G D V K F A E V L E M M G A K V T 300 301 302 303 304 305306 307 308 309 310 311 312 313 314 315 316 317 318 319 Zm W T E T S V TV T G P P R E P F G R K H Zm H6, C1 W T S T S V T V T G P P R E P F G RK H Hel an W T E N S V T V R G P P R N A S G R G H Ory sa W T D T S V TV T G P P R E P Y G K K H Sor ha W T E T S V T V T G P P R Q P F G R K H320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337338 339 Zm L K A I D V N M N K M P D V A M T L A V Zm H6, C1 L K A I D VN M N K M P D V A M T L A V Hel an L R P V D V N M N K M P D V A M T L AV Ory sa L K A V D V N M N K M P D V A M T L A V Sor ha L K A I D V N MN K M P D V A M T L A V 340 341 342 343 344 345 346 347 348 349 350 351352 353 354 355 356 357 358 359 Zm V A L F A D G P T A I R D V A S W R VK Zm H6, C1 V A L F A D G P T A I R D V A S W R V K Hel an V A L Y A D GP T A I R D V A S W R V K Ory sa V A L F A D G P T A I R D V A S W R V KSor ha V A L F A D G P T A I R D V A S W R V K 360 361 362 363 364 365366 367 368 369 370 371 372 373 374 375 376 377 378 379 Zm E T E R M V AI R T E L T K L G A S V E Zm H6, C1 E S E R M V A I R T E L T K L G A SV E Hel an E T E R M I A I C T E L R K L G A T V E Ory sa E T E R M V AI R T E L T K L G A S V E Sor ha E T E R M V A I R T E L T K L G A S V E380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397398 399 Zm E G P D Y C I I T P P E K L N V T A I D Zm H6, C1 E G P D Y CI I T P P G/P K L N V T A I D Hel an E G P D Y C V I T P P E K L N V T AI D Ory sa E G P D Y C I I T P P E K L N I T A I D Sor ha E G P D Y C II T P P E K L N V T A I D 400 401 402 403 404 405 406 407 408 409 410411 412 413 414 415 416 417 418 419 Zm T Y D D H R M A M A F S L A A C AE V P Zm H6, C1 T Y G* D H R M A M A F S L A A C G E V P Hel an T Y D DH R M A M A F S L A A C A D V P Ory sa T Y D D H R M A M A F S L A A C AD V P Sor ha T Y D D H R M A M A F S L A A C A E V P 420 421 422 423 424425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 Zea ma V T IR D P G C T R K T F P D Y F D V L Zm H6, C1 V T I R D P G C T R K T F PD Y F D R L Hel an V T I K D P G C T R K T F P D Y F E V L Ory sa V T IR D P G C T R K T F P N Y F D V L Sor ha V T I R D P G C T R K T F P D YF D V L 440 441 442 443 444 445 Species Genbank # Zea ma S T F V K N Zeamays CAA44974 Zm H6, C1 S Q F V K N 868-H6, 123-C1 Hel an E R F T K HHelianthus anuus Ory sa S T F V R N Oryza sativa AF413082 Sor ha S T F VK N Sorghum halepense H6T5X2 Reverse highlight: H6 and C1 Bold: uniqueto H6 *Asterisk: unique to C1For 10 of the 15 mutations in maize clone 868-H6 (98, 102, 208, 279,302, 361, 391, 402, 416 and 438), the context of the amino acidpositions as well as the chemical character of the native amino acidwere highly homologous. For the remaining amino acid positions (2, 4,72, 84 and 441), the native amino acids in the sunflower sequencediffered significantly from the monocot maize sequence. However, the H6changes were incorporated into the mutated sunflower sequence (SEQ IDNO:10) as indicated in Table 9 regardless of those considerations.

ChloroP Prediction Server (Emanuelsson, O. et al. 1999. Protein Sci.8(5):978-84) was used to approximate the amino terminus of the matureEPSPS proteins. Nucleotide sequences of both native and mutagenizedgenes were optimized for expression in E. coli and synthesized. Thesynthetic genes were cloned into a plasmid vector and expressed,purified and analyzed as described in Example 1. The kinetic parametersof the purified EPSPS proteins are shown in Table 10.

TABLE 10 Kinetic parameters of native EPSPS from various species and thesame enzymes carrying the 868-H6 mutations. K_(M) values are for PEP.k_(cat), min⁻¹ K_(M), uM k_(cat)/K_(M) K_(I), uM k_(cat)/K_(M)*K_(I) Zeama, native 1464 15.7 93.8 0.13 12 (SEQ ID NO: 2) Zea ma, H6 386 24.016.1 629 10070 (SEQ ID NO: 5) Sor ha, native 3056 17.4 176 0.2 33 (SEQID NO: 8) Sor ha H6 317 30.6 10.0 783 8080 (SEQ ID NO: 12) Ory sa,native 1858 12.6 148.0 0.10 18 (SEQ ID NO: 7) Ory sa, H6 293 28.9 10.0907 9200 (SEQ ID NO: 11) Helianthus 1771 18.8 94 0.15 14 annuus, native(SEQ ID NO: 9) Hel an, H6 211 37.2 6.0 1583 8960 (SEQ ID NO: 10)The combination of mutations discovered in 868-H6 maize EPSPS clearlyhad a very similar effect on fitness (k_(cat)/K_(M)*K_(I)) when mappedonto EPSPS from other species. Not surprisingly, the greatest deviationin individual parameters was with sunflower in comparison with themonocot species. Sunflower had higher K_(M) for PEP and a lower k_(cat),resulting in a k_(cat)/K_(M) that was 38% that of maize H6. However, themutant sunflower enzyme had a 74% to 150% higher K_(I) for glyphosatethan the mutant monocot EPSPS enzymes.

Likewise, the mutations present in maize EPSPS 123-C1 (See Table 8) weremapped onto the amino acid sequences of sorghum (SEQ ID NO:23) and theproteins were produced and purified as above. The enzyme activity wasanalyzed by measuring reaction rates when PEP and S3P were both presentat 200 uM, and when substrates were present at 30 uM and glyphosate at 1mM (Table 11).

TABLE 11 Reaction rates of EPSPS enzymes with mutations at G102A aloneor the mutations defined for maize variant 123-C1. Native G102A C1mutations 30 uM, 30 uM, 30 uM, 200 uM, 1 mM 200 uM, 1 mM 200 uM, 1 mMSpecies no glyph glyph no glyph glyph no glyph glyph Sor ha 1376 −2.2239 24.4 356 60.0 Zea ma 1464 0.0 270 41.0 516 94.5For sorghum and maize EPSPS, the benefit of the C1 mutations compared toG102A alone is seen both in the presence and absence of glyphosate, dueto the much lower K_(M) for PEP conferred by the 8 other mutations.

The mutations can also be mapped to other known EPSPS sequences fromvarious crops (see, for example, SEQ ID NOs:13-17 that correspond tomutated versions (see, for example, SEQ ID NOs:18-22) containing the H6mutations). The alignments of the native EPSPS sequences with theircorresponding mutated versions are shown in FIGS. 1-8.

Example 4 Production of Glyphosate-Resistant Maize Expressing GlyphosateTolerant Plant EPSPS

Maize plants expressing EPSPS variant genes are produced using at leasttwo approaches—(i) recombinant DNA-based transformation or site-directedchanges at the endogenous EPSPS genomic locus. Recombinant DNA basedtransformation methods are well known in the art, e.g. Agrobacteriumtumefaciens-mediated and particle bombardment based transformations.

(i) Recombinant Maize EPSPS-Variant Transformation

Agrobacterium tumefaciens based plant transformation vectors areconstructed according to methods known in the art. EPSPS vectors containa T-DNA insert having a constitutive plant promoter, such as anubiquitin promoter, an intron, an optional enhancer such as a 35Senhancer element, an EPSPS variant DNA encoding a glyphosate tolerantEPSPS (e.g., 868-H6), and a plant terminator such as, for example, aPinII terminator. Maize immature embryos are excised and infected withan Agrobacterium tumefaciens vector containing the EPSPS variant ofinterest. After infection, embryos are transferred and cultured inco-cultivation medium. After co-cultivation, the infected immatureembryos are transferred onto media containing 1.0 mM glyphosate. Thisselection generally lasts until actively growing putative transgeniccalli are identified. The putative transgenic callus tissues are sampledusing PCR and optionally a Western assay to confirm the presence of theEPSPS variant gene. The putative transgenic callus tissues aremaintained on 1.0 mM glyphosate selection media for further growth andselection before plant regeneration. At regeneration, callus tissueconfirmed to be transgenic are transferred onto maturation mediumcontaining 0.1 mM glyphosate and cultured for somatic embryo maturation.Mature embryos are then transferred onto regeneration medium containing0.1 mM glyphosate for shoot and root formation. After shoots and rootsemerge, individual plantlets are transferred into tubes with rootingmedium containing 0.1 mM glyphosate. Plantlets with established shootsand roots are transplanted into pots in the greenhouse for furthergrowth, to obtain T0 spray data, and to produce T1 seed.

In order to evaluate the level of glyphosate resistance of thetransgenic maize plants expressing the EPSPS variant transgenes, T0plants are sprayed with glyphosate in the greenhouse. Glyphosateconcentrations include dosage of e.g., 1× rate of a commerciallyavailable glyphosate formulation. Plant resistance levels are evaluatedby plant discoloration scores and plant height measurements. Plantdiscoloration is evaluated according to the following scale:

Discoloration Score at 1, 2, 3 and 4 Weeks After Spray with Glyphosate9=no leaf/stem discoloration7=minor leaf/stem discoloration5=worse leaf/stem discoloration3=severely discolored plant or dying plant1=dead plant

Plant Height Measurements are recorded before spraying with glyphosateand after spraying with glyphosate at 1, 2, 3 and 4 weekspost-application. Two plants are sent to the greenhouse from each event(independent transgenic callus). Plant 1 is kept for seed production andis not sprayed with glyphosate. Plant 2 is sprayed at 2×-4× glyphosate(1× glyphosate=26 ounces/acre) at 14 days after transplanting. The T0plant discoloration scores at 7 and 14 days after the spray are alsoobserved. Height data at tasseling is also measured.

(ii) Guided Cas9-Based EPSPS Modifications

Expression cassettes for guide RNA/Cas endonuclease based genomemodification in maize plants are disclosed at least in Examples 1-15 ofInternational Application No. PCT/US2015/38767, filed Jul. 1, 2015 andherein incorporated by reference.

Described herein is a guide RNA/Cas endonuclease system that is based onthe type II CRISPR/Cas system and includes a Cas endonuclease and aguide RNA (or duplexed crRNA and tracrRNA) that together can form acomplex that recognizes a genomic target site in a plant and introducesa double-strand-break into said target site (U.S. patent application61/868,706, filed Aug. 22, 2013), incorporated herein by reference. Inthis Example, the desired target site is the maize endogenous nativeEPSPS genomic sequence.

The maize optimized Cas9 endonuclease and single guide RNA expressioncassettes containing the specific maize variable targeting domains areco-delivered to e.g., 60-90 Hi-II immature maize embryos byparticle-mediated delivery using techniques well known in the art andoptionally, in the presence of BBM and WUS2 genes (U.S. patentapplication Ser. No. 13/800,447, filed Mar. 13, 2013).

After 7 days, the 20-30 most uniformly transformed embryos are pooledand total genomic DNA is extracted. The region surrounding the intendedtarget site is PCR amplified with Phusion® High Fidelity PCR Master Mix(New England Biolabs, M0531L) adding on the sequences necessary foramplicon-specific barcodes and Illumnia sequencing using “tailed”primers through two rounds of PCR.

The resulting PCR amplifications are purified with a Qiagen PCRpurification spin column; the concentration is measured with a Hoechstdye-based fluorometric assay; the PCR amplifications are combined in anequimolar ratio; and single read 100 nucleotide-length deep sequencingis performed using Illumina's MiSeq Personal Sequencer with a 30-40%(v/v) spike of PhiX control v3 (Illumina, FC-110-3001) to off-setsequence bias. Only those reads with a ≥1 nucleotide indel arisingwithin the 10 nucleotide window centered over the expected site ofcleavage and not found in a similar level in the negative control areclassified as non homologous end-joining mutations. NHEJ mutant readswith the same mutation are counted and collapsed into a single read andthe top 10 most prevalent mutations are visually confirmed as arisingwithin the expected site of cleavage. The total numbers of visuallyconfirmed NHEJ mutations are then used to calculate the % mutant readsbased on the total number of reads of an appropriate length containing aperfect match to the barcode and forward primer.

The frequency of NHEJ mutations recovered by deep sequencing for theguide RNA/Cas endonuclease system targeting the one or more desiredEPSPS targets (e.g., one or more mutations of the 868-H6 variant)compared to the cas9 only control is analyzed. This Example describesthat the guide RNA/Cas9 endonuclease system described herein can be usedto introduce a double strand break at genomic sites of interest withinthe maize endogenous EPSPS genomic regions. Editing the EPSPS targetresults in the production of plants that are tolerant and/or resistantagainst glyphosate based herbicides.

Example 5 Efficacy of Shuffled Plant EPSPS for Conferring GlyphosateTolerance in Transformed Plants

Transformation vectors were constructed consisting of nucleotidesequences coding for either the native maize EPSPS or maize EPSPSvariant H6 (the nucleotide sequences are SEQ ID NO:24 and SEQ ID NO:25,respectively). Each was preceded by nucleotide sequences coding foreither an Arabidopsis chloroplast targeting peptide (SEQ ID NO:26) or anartificial CTP termed 6H1 (U.S. Pat. No. 7,345,143; SEQ ID NO:27). Theresulting four CTP-enzyme combinations were preceded either by thenative Arabidopsis EPSPS promoter (AT1G48860; SEQ ID NO:28), theubiquitin-3 promoter (SEQ ID NO:29), or the ubiquitin-10 promoter(Norris et al. 1993. Plant Mol Biol 21:895-906; SEQ ID NO:30) for atotal of 12 combinations of promoter, CTP and enzyme. A polynucleotidecoding for the hemagglutinin affinity tag (nucleotide sequence is SEQ IDNO:31) was fused to the C-terminus of each EPSPS coding region, followedby a phaseolin terminator (SEQ ID NO:32).

Binary vectors for Agrobacterium-mediated transformation wereconstructed using standard molecular biology techniques. Arabidopsisthaliana Col-0 transformation was carried out using a modified floraldip method (Clough and Bent. 1998. Plant J 16:735-743), in which theflowering parts of the plant are dipped into a suspension ofAgrobacterium tumifaciens strain GV3101 transformed with the 12different binary vectors, designated PHD6020-PHD6031, as described inTable 12.

TABLE 12 Description of the binary vectors used for Arabidopsistransformation pHD# Vector Description pHD6020 OriPUC::UBQ3 PRO::NativeEPSPS CTP::MzWT CDs::HA Cterm tag::KanR PHD6021 OriPUC::UBQ3 PRO::NativeEPSPS CTP::Hit H6 CDs::HA Cterm tag::KanR pHD6022 OriPUC::UBQ10PRO::Native EPSPS CTP::MzWT CDs::HA Cterm tag::KanR pHD6023OriPUC::UBQ10 PRO::Native EPSPS CTP::Hit H6 CDs::HA Cterm tag::KanRpHD6024 OriPUC::Native PRO::Native EPSPS CTP::MzWT CDs::HA Ctermtag::KanR pHD6025 OriPUC::Native PRO::Native EPSPS CTP::Hit H6 CDs::HACterm tag::KanR pHD6026 OriPUC::UBQ3 PR0::6H1 CTP::MzWT CDs::HA Ctermtag::KanR pHD6027 OriPUC::UBQ3 PR0::6H1 CTP::Hit H6 CDs::HA Ctermtag::KanR pHD6028 OriPUC::UBQ10 PR0::6H1 CTP::MzWT CDs::HA Ctermtag::KanR pHD6029 OriPUC::UBQ10 PR0::6H1 CTP::Hit H6 CDs::HA Ctermtag::KanR pHD6030 OriPUC::Native PR0::6H1 CTP::MzWT CDs::HA Ctermtag::KanR pHD6031 OriPUC::Native PR0::6H1 CTP::Hit H6 CDs::HA Ctermtag::KanR

After inoculation, the plants were placed in a plant growth chamber setfor a 16 hr photoperiod. Conditions by day were 21° C. with a lightintensity of 280 μM/m2/s and by night, 18° C. Seeds were collected afterthe pods turned to brown. Seeds were surface sterilized with 95% ethanolfor 1 minute, then in 20% bleach plus one drop of Tween-20 for 15minutes and washed 3 times with the sterile water. Thirty mg ofsterilized seed were plated on the agar selection medium, composed of MSsalts with vitamins (e.g., SigmaAldrich, M0404), 1% sucrose, 8% TC Agar,100 mg/L Timentin and 50 mg/L kanamycin at pH 5.7 in 150×25 mm petridishes (e.g., Falcon Large Petri Dishes, VWR Cat #351013). Plates weresealed with parafilm and incubated at 21° C., 16 hour photoperiod at60-100 μE/m2/s for germination and growth. Events that survived theselection were transplanted to RediEarth potting soil (SunGro) and grownin a growth chamber (16 hr photoperiod, with day conditions at 21° C.with a light intensity of 280 μM/m2/s and 18° C. at night). Twenty twodays after transplanting, plants were sprayed with Touchdown at rates of0.42, 0.84 or 1.26 kg ai/ha (0.5, 1.0 and 1.5 times the standard fieldapplication rate, respectively). Plants were evaluated for injury andphenotype 6 and 10 days after treatment.

At the 1.26 kg/Ha spray rate, untransformed plants did not grow at allafter treatment and by 10 days, exhibited chlorosis and imminentnecrosis. With either the Arabidopsis native EPSPS or the synthetic 6H1CTP, tolerance to glyphosate correlated with the strength of thepromoter (UbiQ10>UbiQ3>native). Plants transformed with constructscontaining the UbiQ10 promoter and the H6 variant had no visible injuryor growth inhibition compared to unsprayed controls. Although nativeEPSPS conferred some tolerance with the stronger promoters, the improvedfitness ([kcat/KM]*Ki) of the H6 variant is clearly seen at everycondition.

Example 6 Efficacy of Shuffled Plant EPSPS for Conferring GlyphosateTolerance in Transformed Soybean

Transformation vectors were constructed consisting of nucleotidesequences coding for either the native maize EPSPS, the maize EPSPSvariant H6, the maize EPSPS variant C1, or the maize EPSPS variant C2(the nucleotide sequences are provided as SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:33, or SEQ ID NO:34, respectively). Each was preceded bynucleotide sequences coding for an artificial CTP termed 6H1 (U.S. Pat.No. 7,345,143; SEQ ID NO:27). The resulting CTP-enzyme combinations werepreceded either by the native Arabidopsis EPSPS promoter (AT1G48860; SEQID NO:28), the ubiquitin-3 promoter (SEQ ID NO:29), or the ubiquitin-10promoter (Norris et al. 1993, supra; SEQ ID NO:30). A polynucleotidecoding for the hemagglutinin affinity tag (nucleotide sequence is SEQ IDNO:31) was fused to the C-terminus of each EPSPS coding region, followedby a phaseolin terminator (SEQ ID NO:32).

Binary vectors for Agrobacterium mediated transformation wereconstructed using standard molecular biology techniques. Glycine max(93Y21) hairy root transformation was carried out using a methodslightly modified from that of Cho et al. (Cho et al. 2000. Planta210:195-204), in which the wounded cotyledon explants were infected witha suspension of Agrobacterium rhizogenes strain K599 transformed withthe binary vectors described in Table 13.

TABLE 13 Description of the binary vectors used for Arabidopsistransformation pHD# Vector Description pHD6026 OriPUC::UBQ3 PR0::6H1CTP::MzWT CDs::HA Cterm tag:: Kan R pHD6027 OriPUC::UBQ3 PR0::6H1CTP::Hit H6 CDs::HA Cterm tag:: Kan R pHD6028 OriPUC::UBQ10 PR0::6H1CTP::MzWT CDs::HA Cterm tag:: Kan R pHD6029 OriPUC::UBQ10 PR0::6H1CTP::Hit H6 CDs::HA Cterm tag:: Kan R pHD6030 OriPUC::Native PR0::6H1CTP::MzWT CDs::HA Cterm tag:: Kan R pHD6031 OriPUC::Native PR0::6H1CTP::Hit H6 CDs::HA Cterm tag:: Kan R pHD5766 OriPUC::UBQ3 PR0::6H1CTP::HitC1 CDs::HA Cterm tag:: Kan R pHD5767 OriPUC:: UBQ3 PRO::, 6H1CTP::Hit C2 CDs::HA Cterm tag:: Kan R pHD5768 OriPUC::UBQ10 PRO::6H1CTP::Hit C1 CDs::HA Cterm tag:: Kan R pHD5769 OriPUC::UBQ10 PRO::6H1CTP::Hit C2::HA Cterm tag:: Kan R pHD5770 OriPUC::Native PRO::6H1CTP::Hit C1 CDs::HA Cterm tag:: Kan R pHD5771 OriPUC::Native PRO::6H1CTP::Hit C2::HA Cterm tag:: Kan R

After co-cultivation, the explants were placed onto growth mediumcontaining kanamycin for selection of transformation events, with orwithout 25 uM glyphosate. Untransformed cotyledons formed a dense growthof roots on medium lacking glyphosate, but no growth on the same mediumcontaining 25 uM glyphosate (not shown). Cotyledons transformed with allconstructs formed dense root growth, indistinguishable from that seenwith untransformed cotyledons, on medium lacking glyphosate (FIG. 9).Hairy roots were not formed from cotyledons transformed with constructswhere the gene coding for EPSPS was driven by the weaker promoters (UBQ3and native). However, cotyledons transformed with constructs containingthe UBQ10 promoter and the H6, C1 and C2 variants generated hairy rootsin the presence of 25 uM glyphosate, while the native EPSPS supported noroot growth.

Example 7 Endogenous Genome Editing of EPSPS Gene Locus

Maize optimized Cas9 endonucleases are developed and evaluated for theirability to introduce one or more double-strand breaks at the EPSPSgenomic target sequence. A maize optimized Cas9 endonuclease (moCas9) isgenerally supplemented with a nuclear localization signal (e.g., SV40)by adding the signal to the 5′ end of the moCas9 coding sequence. Theplant moCas9 expression cassette is subsequently modified by insertionof an intron into the moCas9 coding sequence in order to enhance itsexpression in maize cells and to eliminate its expression in E. coli andAgrobacterium. The maize ubiquitin promoter and the potato proteinaseinhibitor II gene terminator sequences complement the moCas9endonuclease gene designs. However, any other promoter and/or terminatorcan be used.

A single guide RNA (sgRNA) expression cassette includes for example, U6polymerase III maize promoter and its cognate U6 polymerase IIItermination sequences. The guide RNA includes a nucleotide variabletargeting domain followed by a RNA sequence capable of interacting withthe double strand break-inducing endonuclease.

A maize optimized Cas9 endonuclease target sequence (moCas9 targetsequence) within the EPSPS codon sequence is complementary to thenucleotide variable sequence of the guide sgRNA, which determines thesite of the Cas9 endonuclease cleavage within the EPSPS coding sequence.This targeting region can vary based on the nature and the number ofmutations to be targeted within the EPSPS locus.

The moCAS9 target sequence is synthesized and cloned into the guideRNA-Cas9 expression vector designed for delivery of the components ofthe guide RNA-Cas9 system to the maize cells throughAgrobacterium-mediated transformation. Agrobacterium T-DNA also deliversthe yeast FLP site-specific recombinase and the WDV (wheat dwarf virus)replication-associated protein (replicase), if needed. If the moCas9target sequences are flanked by the FLP recombination targets (FRT),they can be excised by FLP in maize cells forming episomal(chromosome-like) structures. Such circular DNA fragments are replicatedby the WDV replicase (the origin of replication was embedded into theWDV promoter) allowing their recovery in E. coli cells. If the maizeoptimized Cas9 endonuclease makes a double-strand break at the moCas9target sequence, its repair might produce mutations. The procedure isdescribed in detail in: Lyznik, L. A., Djukanovic, V., Yang, M. andJones, S. (2012) Double-strand break-induced targeted mutagenesis inplants. In: Transgenic plants: Methods and Protocols (Dunwell, J. M. andWetten, A. C. eds). New York Heidelberg Dordrecht London: Springer, pp.399-416. The maize optimized Cas9 endonuclease described herein isfunctional in maize cells and efficiently generates double-strand breaksat the moCas9 target sequence.

In order to accomplish targeted genome editing of the maize chromosomalEPSPS gene, a polynucleotide modification template for editing the EPSPScoding sequence may be created and co-delivered with the guide RNA/Cas9system components. There can be more than one modification templatedelivered simultaneously or sequentially.

A polynucleotide modification template includes one or more nucleotidemodifications (e.g., nucleotide changes that correspond to the one ormore amino acid changes disclosed herein) when compared to the nativeEPSPS genomic sequence to be edited. These nucleotide modifications aregenerally substitution mutations. The EPSPS template sequences mayencode a functional EPSPS protein or may be partial fragments that donot encode a full-length functional polypeptide.

The EPSPS polynucleotide modification template may be co-delivered withthe guide sgRNA expression cassette and a maize optimized Cas9endonuclease expression vector, which contains the maize optimized Cas9endonuclease expression cassette and a selectable marker gene, usingparticle bombardment. Ten to eleven day-old immature embryos are placedembryo-axis down onto plates containing N6 medium and are incubated at28° C. for 4-6 hours before bombardment. The plates are placed on thethird shelf from the bottom in the PDS-1000 apparatus and bombarded at200 psi. Post-bombardment, embryos are incubated in the dark overnightat 28° C., transferred to plates containing N6-2 media, and then storedfor 6-8 days at 28° C. The embryos are then transferred to platescontaining N6-3 media for three weeks. Responding callus is thentransferred to plates containing N6-4 media for an additional three-weekselection. After six total weeks of selection at 28° C., a small amountof selected tissue is transferred onto the MS regeneration medium andincubated for three weeks in the dark at 28° C.

Multiple callus events selected on media containing appropriatesubstrate for the selectable marker (e.g., bialophos for the moPATselectable marker gene) are screened for the presence of the targetedpoint mutations. Further sequencing of the EPSPS locus is performed toconfirm the mutations. Plantlets are generated from the callus eventsfollowing standard procedures.

Example 8 Rapid High-Throughput Enzyme Assay for Multiple EnzymeVariants in the Presence of Inhibitor

One of the commercial applications of directed evolution is todesensitize an enzyme to inhibition by, for example, a herbicide. kcat,1/K_(M) and K_(I) are three dimensions that when multiplied are ameasure of an enzyme's intrinsic capacity for catalysis in the presenceof an inhibitor. When attempting to optimize those values by directedevolution, (k_(cat)/K_(M))*K_(I) can be an informative parameter forevaluating libraries of variants. However, evaluating(k_(cat)/K_(M))*K_(I) for hundreds of variants by substrate saturationanalysis may not provide adequate throughput. Manipulation of theMichaelis-Menten equation that enables isolation of(k_(cat)/K_(M))*K_(I) on one side of the equation is one approach toexpedite the throughput of the assays. If substrate and enzymeconcentrations are identical but velocity is measured at two differentinhibitor concentrations (one of which can be 0), this Exampledemonstrates that the data are sufficient to calculate(k_(cat)/K_(M))*K_(I) with just two rate measurements. The procedure isvalidated by correlating values obtained with the rapid method withthose obtained by substrate saturation kinetics.

Directed evolution is a process for improving an enzyme's fitness in aproperty defined by a commercial or academic interest, directed byempirical observations of the fitness of variants generated in vitro. Inthe case where the goal is to desensitize the enzyme to an inhibitor(e.g., herbicide or feedback-inhibiting metabolite), the improvementwill be made through elevating the value of KI, the dissociationconstant of the enzyme-inhibitor complex, as shown in Scheme 1:

Rarely will an increase in KI come without affecting the otherparameters, so some measurement that captures all three parameters ispreferred to be used. The parameter (k_(cat)/K_(M))*K_(I) combines anexpression of catalytic efficiency (k_(cat)/K_(M)) with one of affinityfor inhibitor compared to substrate (KI/KM). Improved(k_(cat)/K_(M))*K_(I) can be attained by increased k_(cat), decreasedK_(M), increased K_(I) or any combination. Increasing kcat is moreeffective than reducing K_(M):

$\begin{matrix}{v_{i} = \frac{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}{{K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)} + \lbrack S\rbrack}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Increasing K_(I) is effective until it reaches the approximate inhibitorconcentration, after which further increases will proportionatelyincrease (k_(cat)/K_(M))*K_(I), but can only result in a further 2-foldincrease in v_(i).

Direct evaluation of a library of variants is performed by measuringreaction velocity under conditions of substrate and inhibitorconcentration, pH and ionic strength, if known. The rate obtained isdescribed by Equation 1. If at the outset of the enzyme improvementproject, velocity is set at application conditions as the sole criterionfor improvement, one risks becoming locked into a sequence context thatleads to a peak separated from a much higher potential maximum. One canavoid descending all the way to the bottom by having an alternativefitness parameter, (k_(cat)/K_(M))*K_(I), that captures variants thatare improved in one or two of the individual parameters. Monitoring(k_(cat)/K_(M))*K_(I) has the added benefit of revealing whether moreoptimization could be attained given a more favorable distribution ofthe values of the individual parameters. For example, a variant that hasa v_(i) under application conditions that is on par with the currentfittest variants could have a K_(I) sufficiently high that(k_(cat)/K_(M))*K_(I) is two or more fold greater than the othercandidates. The value of such a variant can be seen with some samplecalculation in the Michaelis-Menten equation. If K_(I) is 5000 uM andK_(M) 100 uM, and if the concentrations of I and S are 1000 uM and 20uM, respectively, the denominator in the rate equation is 140. However,if through further mutagenesis, the K_(I) and K_(M) were reducedproportionately (e.g., 5-fold) to 1000 and 20 uM respectively, thedenominator would be 60 and v_(i) would increase by 2.33-fold (140/60).Thus, (k_(cat)/K_(M))*K_(I) can be a useful adjunct to measuring v_(i)under application conditions for guiding directed evolution forinsensitivity to an inhibitor.

Generally, (k_(cat)/K_(M))*K_(I) is obtained by performing substratesaturation analysis in the absence and presence of inhibitor. However,that analysis takes longer. Therefore, a novel treatment of theMichaelis-Menten equation for competitive inhibition that enablesaccurate estimation of (k_(cat)/K_(M))*K_(I) with just two ratemeasurements significantly increases the throughput and expedites thescreening process for evaluating hundreds and thousands of variants. Tovalidate the method, (k_(cat)/K_(M))*K_(I) was quantified using bothmethods—traditional (saturation) and the instant method (rapid), forvariants of maize 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)and its competitive inhibitor, glyphosate.

In the Michaelis-Menten equation for steady state reaction velocity withcompetitive inhibition, the term (k_(cat)/K_(M))*K_(I) cannot beisolated due to the K_(M) and [S] terms in the denominator. However, iftwo rate measurements are made at different inhibitor concentrations,those terms can be eliminated by subtraction and (kcat/KM)*KI isolated,as follows:

$\begin{matrix}{{\upsilon_{i} = {\frac{V_{\max}\lbrack S\rbrack}{{K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)} + \lbrack S\rbrack} = \frac{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}{{K_{m}\left( {1 + \frac{\lbrack I\rbrack}{K_{i}}} \right)} + \lbrack S\rbrack}}}\begin{matrix}{{\frac{1}{\upsilon_{1\; i}} - \frac{1}{\upsilon_{i\; 2}}} = {\frac{{K_{m}\left( {1 + \frac{\lbrack I\rbrack_{1}}{K_{i}}} \right)} + \lbrack S\rbrack}{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack} - \frac{{K_{m}\left( {1 + \frac{\lbrack I\rbrack_{2}}{K_{i}}} \right)} + \lbrack S\rbrack}{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}}} \\{= \frac{K_{m} + {K_{m}\frac{\lbrack I\rbrack_{1}}{K_{i}}} + \lbrack S\rbrack - K_{m} - {K_{m}\frac{\lbrack I\rbrack_{2}}{K_{i}}} - \lbrack S\rbrack}{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}} \\{= \frac{{K_{m}\frac{\lbrack I\rbrack_{1}}{K_{i}}} - {K_{m}\frac{\lbrack I\rbrack_{2}}{K_{i}}}}{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}} \\{= \frac{\frac{K_{m}}{K_{i}}\left( {\lbrack I\rbrack_{1} - \lbrack I\rbrack_{2}} \right)}{{k_{cat}\lbrack E\rbrack}\lbrack S\rbrack}} \\{= {\frac{K_{m}}{k_{cat}K_{i}} \times \frac{\lbrack I\rbrack_{1} - \lbrack I\rbrack_{2}}{\lbrack E\rbrack \lbrack S\rbrack}}}\end{matrix}\begin{matrix}{{\frac{k_{cat}}{K_{m}} \times K_{i}} = {\frac{1}{\frac{1}{\upsilon_{1\; i}} - \frac{1}{\upsilon_{i\; 2}}} \times \frac{\lbrack I\rbrack_{1} - \lbrack I\rbrack_{2}}{\lbrack E\rbrack \lbrack S\rbrack}}} \\{= {\frac{\upsilon_{1\; i} \times \upsilon_{i\; 2}}{\upsilon_{i\; 2} - \upsilon_{1\; i}} \times \frac{\lbrack I\rbrack_{1} - \lbrack I\rbrack_{2}}{\lbrack E\rbrack \lbrack S\rbrack}}}\end{matrix}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

where v₁ and v₂ are initial velocities at identical substrate ([S]) andenzyme ([E]) concentrations, but at two inhibitor concentrations, [I]₁and [I]₂. Furthermore, when [I]₂=0, equation 2 can be simplified to

$\begin{matrix}\begin{matrix}{{\frac{k_{cat}}{K_{m}} \times K_{i}} = {\frac{1}{\frac{1}{\upsilon_{i}} - \frac{1}{\upsilon_{0}}} \times \frac{\lbrack I\rbrack}{\lbrack E\rbrack \lbrack S\rbrack}}} \\{= {\frac{\upsilon_{i} \times \upsilon_{0}}{\upsilon_{0} - \upsilon_{i}} \times \frac{\lbrack I\rbrack}{\lbrack E\rbrack \lbrack S\rbrack}}}\end{matrix} & {{Equation}\mspace{14mu} 3}\end{matrix}$

where v₀ and v₁ are initial velocities without and with inhibitor but atthe same substrate and enzyme concentrations. Although Equations 2 and 3are equally valid, to generate the data in Table 1, rate measurement wasperformed with and without inhibitor using Equation 3.

Source of Reagents, Enzyme and Variants

Shikimate-3-phosphate (S3P) was prepared from cultures of Klebsiellapneumonia aroA-(ATCC 25597). Cells from a 500 ml culture grown in 2×YTwere used to inoculate 6 L of minimal medium augmented with 55 uMtyrosine, 60 uM phenylalanine, 25 uM tryptophan, 0.1 uM 4-aminobenzoateand 0.1 uM 4-hydroxybenzoate (Weiss et al., 1953. J Amer Chem Soc75:5572-5576). Accumulation of S3P was monitored by anion exchange HPLC.After about 4 days shaking at 37 C, the concentration reached ˜1 mM. S3Pwas purified from the culture supernatant by anion exchangechromatography in ammonium bicarbonate at pH 7.3, with gradient elutionup to 0.7 M. S3P was cleanly separated from phosphate, which elutedearlier. 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG) wasfrom Setareh Biotech, Eugene Oreg. All other reagents were fromSigma-Aldrich.

The amino acid sequence of mature Zea mays EPSPS was obtained fromGenBank entry CAA44974.1 (SEQ ID NO:2). A nucleotide sequence wascreated to add an N-terminal methionine and to optimize codon usage forexpression in E. coli. The synthesized gene was cloned into anexpression vector that provides a T7 promoter driving expression of theprotein and a 10× N-terminal histidine tag. Variants selected for thisstudy include native maize EPSPS and variants generated by a geneshuffling cascade described herein. The proteins were expressed in E.coli BL21(DE3) and purified by Ni-NTA resin (Qiagen). Proteinconcentration was determined by absorbance at 280 nm using an extinctioncoefficient of 0.676 OD/mg/m L. The proteins were normalized to 0.5mg/mL for assay.

Enzyme Assay Procedure and Data Analysis

EPSPS catalyzes the following reaction: Phosphoenolpyruvate(PEP)+3-phosphoshikimate (S3P)=phosphate(Pi)+5-enolpyruvylshikimate-3-phosphate (EPSP). EPSPS activity wasdetermined by quantifying the phosphate generated by the reaction.Release of phosphate was coupled to reaction with MESG, catalyzed bypurine-nucleoside phosphorylase, using standard methods. The absorbancechange that occurs was monitored continuously at 360 nm, where theextinction is 11,200 M-1 cm-1, with a Spectramax plate reader (MolecularDevices). To determine kinetic parameters in the conventional way, PEPwas present at seven concentrations (the eighth being the blank,containing no substrate) ranging from 15 to 800 uM and the unvariedsubstrate S3P was present at the saturation concentration of 200 uM.Five microliters of 60-fold concentrated stock solutions of PEP wereplaced in the wells of the 96-well assay plate and reactions werestarted with the addition of a mixture containing 25 mM Hepes, pH 7, 100mM KCl, 5% (v/v) ethylene glycol, 0.2 mM MESG, 1 unit/ml purinenucleoside phosphorylase (Sigma N8264), 200 uM S3P and EPSPS. The enzymeconcentration was adjusted so as to generate sufficient signal withoutexceeding the limit for linear initial reaction rates. The sameprocedure was repeated with two or three concentrations of glyphosateand the data were processed by non-linear regression analysis withGraphPad Prism (graphpad.com) and globally fitted to theMichaelis-Menten equation for competitive inhibition.

For the novel method, the identical assay conditions were used. The PEPconcentration was set at 30 uM, which is close to the K_(M) of wild-typeEPSPS, while S3P was present at 200 uM. The concentration of EPSPS wasfixed at 0.07 uM. Reactions were performed in triplicate, with orwithout 1 mM glyphosate as inhibitor. Values for v₀ and v₁ were enteredinto equation 3, yielding (k_(cat)/K_(M))*K_(I).

To validate that the rapid method yields an accurate estimation of(k_(cat)/K_(M))*K_(I), the results were compared with those obtained byfull substrate saturation analysis. The data were obtained for nativeand shuffled variants of maize 5-enolpyruvylshikimate-3-phosphate (EPSP)synthase. To establish a correlation between actual and surrogate(k_(cat)/K_(M))*K_(I), a panel of variants was selected exhibiting awide range of known individual parameters for analysis by the rapidmethod. Native maize and another EPSPS formed the low and high end ofthe range. Values in between were supplied by Zm-H6 EPSPS and singlemutations thereof. Zm-H6 EPSPS was generated by gene shuffling of nativemaize EPSPS and has 16 mutations relative to the native enzyme, asdescribed herein. The kinetic parameters of the variants spanned a rangeof 39-fold for kcat, 37-fold for KM, 10000-fold for KI and 9000-fold for(k_(cat)/K_(M))*K_(I). All parameters obtained by both methods are shownin Table 15. Linear regression of the values for (k_(cat)/K_(M))*K_(I)determined by substrate saturation analysis and the rapid method showsan excellent correlation throughout the range of values (FIG. 10).

With two simple reactions, a lumped parameter (k_(cat)/K_(M))*K_(I) isgenerated that captures the kinetic properties essential for catalysisin the presence of inhibitor. Given an appropriate assay, the tworeactions could be performed with automated liquid handling, enablingevaluation of hundreds of variants. These measurements would accompany arate measurement at the conditions of the application using the sameautomated assay.

When inhibition is involved, multiplying (k_(cat)/K_(M)) by K_(I)additionally captures the magnitude of the inhibitor's dissociationconstant. The first steps would be rate measurement at applicationconditions and the two measurements for determining(k_(cat)/K_(M))*K_(I) by the rapid method. Next, the values for(k_(cat)/K_(M))*K_(I) of the entire lot are compared with those of thebest variants as determined by the criterion of rate at applicationconditions. If values for (k_(cat)/K_(M))*K_(I) stand out over thosepossessed by the best variants under application conditions, itindicates that exceptional individual parameters are present within thepopulation and that further optimization is possible. Any associationbetween outstanding individual parameters and specific mutations maysuggest strategies through which the best individual parameters can becaptured in one enzyme. Thus, beneficial mutations that could contributeto improved performance in later rounds of shuffling could be missedwhen relying on rate at application conditions as the sole screeningcriterion. This is illustrated by the hypothetical data provided inTable 14.

TABLE 14 Hypothetical Kinetic Data for Enzyme Variants Variant kcat KMkcat/KM KI (k_(cat)/K_(M))*K_(I) v_(i) A 500 25 20.0 400 8000 93 B 30075 4.0 7000 28000 57 C 300 15 20.0 1000 20000 120 D 1500 75 20.0 100020000 177Application condition: [S], 20 uM; [I], 1000 uM; [E], 1 uMVariant A is a better variant than variant B in terms of catalyticperformance under the application condition (v_(i)), even though variantB has much greater (kcat/KM)*KI, due solely to its 17.5-fold higher KI.As explained above, values of KI above the [I] have a diminishing effecton v_(i), reaching at most 2-fold. However, this property couldpotentially be exchanged for a lower KM (Variant C) or higher kcat(Variant D) in later rounds of shuffling to eventually obtain a varianthaving better performance under application conditions than any of itsparents.

There are several practical considerations for accurately estimating(k_(cat)/K_(M))*K_(I) with the rapid method. 1) An enzyme concentrationmust be found that yields linear initial reaction rates both with andwithout inhibitor. 2) The inhibitor concentration must be adjusted so asto obtain a degree of inhibition that minimally amplifies the error inthe term v₀-v_(i) in Equation 3. If inhibition is too little, v₀-v_(i)will be small, and the error in the multiplier v_(i)×v₀/(v₀-v_(i)) willbe large. 50% inhibition was set as the target. 3) Substrateconcentration should be set at the approximate K_(M) of the parentalvariant(s), subject to the sensitivity of the assay. High substrateconcentration obscures sensitivity to the inhibitor and reducesstringency for capturing improvements in K_(M). Depending on thenecessity for speed, there exists opportunity for customizing enzyme andinhibitor concentrations. Table 15 shows that the conditions selected,0.07 uM enzyme and 1 mM glyphosate, were inappropriate for accurateanalysis of some of the variants. The rapid method was repeated forthose and obtained data that correlated better with data obtained bysubstrate saturation analysis. For screening purposes however, thatcorrelation step is not necessary. Conditions can be set so thatvariants with a pre-determined minimal fitness level are accuratelyquantified.

In Table 15, parameters above the heavy line for the rapid method wereobtained under standard conditions. For variants shown below the heavyline, the rapid analysis was repeated with enzyme or glyphosateconcentrations adjusted as needed to generate sufficient signal withinthe limits for linear initial rates.

Thus, in summary, this Example demonstrates that (k_(cat)/K_(M))*K_(I)can be determined with just two rate measurements to evaluate pluralityof enzyme variants. Because it quantifies the intrinsic capacity forcatalysis in the presence of an inhibitor, (k_(cat)/K_(M))*K_(I)captures variants with mutations whose properties may be incorporated insubsequent rounds of optimization. Because of its inclusion of K_(M) asa parameter subject to improvement, the method is also suited to in vivoapplications, where there is no control over substrate concentration.

TABLE 15 (k_(cat)/K_(M))*K_(I) for variants of EPSPS determined by therapid method and by substrate saturation kinetic analysis Rapid methodSubstrate saturation EPSPS [E], [Gly], v₀, v_(i), k_(cat)*K_(I) kcat,Km, Ki, k_(cat)*K_(I) variant uM uM uM/min uM/min K_(M) min⁻¹ uM uMK_(M) Zm-native 0.07 1000 43.74 0 n/a 1036 11.0 0.13 12 Bacterial 0.071000 40.11 30.92 64262 1219 15.8 1412 108900 EPSPS Zm-T103A 0.07 10004.28 0.90 543 451 292 208 321 Zm-A189T 0.07 1000 2.30 0.65 431 258 307337 283 Zm-T103I 0.07 1000 3.67 0.26 133 320 171 53 99 Zm-P107L 0.071000 22.35 0.23 111 1376 59.7 3.05 70 Zm-P107S 0.07 1000 35.15 0.15 721798 14.8 0.42 51 Zm-G102A 0.07 1000 4.45 2.80 3596 859 407 1336 2820Zm-H6 0.07 1000 6.92 3.80 4013 198 20.6 381 3660 H6-H54E 0.07 1000 8.054.36 4529 248 27.9 488 4340 H6-A36G 0.07 1000 7.79 4.27 4500 246 30.8454 3630 H6-V87T 0.07 1000 5.60 3.05 3190 259 27.5 348 3280 H6-A76V 0.071000 4.66 2.43 2418 209 45.2 538 2490 H6-K246G 0.07 1000 6.99 3.58 3495210 24.0 284 2490 H6-A69V 0.07 1000 6.07 3.08 2977 161 21.1 320 2440H6-D196V 0.07 1000 2.30 1.40 1704 164 61.8 699 1860 H6-R61Y 0.07 10004.84 2.18 1889 125 23.6 309 1640 H6-Q143E 0.07 1000 2.42 1.35 1454 15046.8 493 1580 H6-A288G 0.07 1000 2.34 1.08 955 60 20.3 443 1310 H6-A185G0.07 1000 1.44 0.78 810 46 21.3 339 732 Zm native 0.007 0.5 6.73 2.75 111036 11.0 0.13 12 CP4 0.007 2000 6.81 4.31 111800 1219 15.8 1412 108900Zm-T103I 0.103 100 5.47 2.16 116 320 171 53 99 Zm-P107L 0.014 10 6.342.39 91 1376 59.7 3.05 70 Zm-P107S 0.007 5 6.41 1.34 40 1798 14.8 0.4251

1.-27. (canceled)
 28. A polynucleotide construct that provides a guideRNA in a plant cell, wherein the guide RNA targets an endogenous EPSPsynthase (EPSPS) gene of the plant cell, wherein the guide RNA as partof a CRISPR complex generates a modified endogenous EPSPS gene thatencodes a plant EPSPS polypeptide that comprises G102A and at least oneamino acid mutation selected from the group consisting of: a) A2R, b)A4W, c) H54M, d) A72Q, e) K84R, f) L98C, g) K173R, h) I208L, i) K243E,j) T279A, k) E302S, l) T361S, m) E391P, n) E391G, o) D402G, p) A416G, q)V438R, r) S440R, s) T441Q, and t) F442V, wherein each amino acidmutation position corresponds to the amino acid position set forth inSEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes apolypeptide comprising a sequence that is at least 90% identical to SEQID NO:2.
 29. The construct of claim 28, wherein said polynucleotideconstruct comprises one or more polynucleotide modification templates togenerate a modified endogenous EPSPS gene that encodes a plant EPSPSpolypeptide with at least two of the amino acid mutations selected fromthe group consisting of a)-t).
 30. The construct of claim 28, whereinsaid polynucleotide construct comprises one or more polynucleotidemodification templates to generate a modified endogenous EPSPS gene thatencodes a plant EPSPS polypeptide comprising A4W, H54M L98C, G102A,K173R I208L, K243E E302S, T361S, E391P, D402G, A416G, V438R, S440R,T441Q, and F442V.
 31. The construct of claim 28, wherein saidpolynucleotide construct comprises one or more polynucleotidemodification templates to generate a modified endogenous EPSPS gene thatencodes a plant EPSPS polypeptide comprising A2R, A4W, A72Q, K84R, L98C,G102A, I208L, T279A, E302S, T361S, E391G, D402G, A416G, V438R, andT441Q.
 32. The construct of claim 28, wherein said polynucleotideconstruct comprises one or more polynucleotide modification templates togenerate a modified endogenous EPSPS gene that encodes a plant EPSPSpolypeptide comprising A2R, A4W, K84R, L98C, K208L, K243E, E391P, andD402G.
 33. The construct of claim 28, wherein said polynucleotideconstruct comprises one or more polynucleotide modification templates togenerate a modified endogenous EPSPS gene that encodes a plant EPSPSpolypeptide having the amino acid sequence set forth in SEQ ID NO:4, SEQID NO:5, or SEQ ID NO:6.
 34. A method for producing a glyphosatetolerant plant, the method comprising: a) providing a guide RNA, atleast one polynucleotide modification template, and at least one Casendonuclease to a plant cell, wherein the at least one Cas endonucleaseintroduces a double strand break at an endogenous EPSP synthase (EPSPS)gene in the plant cell, and wherein said polynucleotide modificationtemplates are used to generate a modified EPSPS gene that encodes aplant EPSPS polypeptide that comprises G102A and at least one amino acidmutation selected from the group consisting of i. A2R, ii. A4W, iii.H54M, iv. A72Q, v. K84R, vi. L98C, vii. K173R, viii. I208L, ix. K243E,x. T279A, xi. E302S, xii. T361S, xiii. E391P, xiv. E391G, xv. D402G,xvi. A416G, xvii. V438R, xviii. S440R, xix. T441Q, and xx. F442V,wherein each amino acid mutation position corresponds to the amino acidposition set forth in SEQ ID NO:1 and wherein the endogenous plant EPSPSgene encodes a polypeptide comprising a sequence that is at least 90%identical to SEQ ID NO:2; b) obtaining a plant from the plant cell of(a); and d) generating a glyphosate tolerant progeny plant that is voidof said guide RNA and Cas endonuclease from the plant of (b).
 35. Themethod of claim 34, wherein the at least one polynucleotide modificationtemplate generates a modified endogenous EPSPS gene encoding a plantEPSPS polypeptide with at least two of the amino acid mutations selectedfrom the group consisting of a)-t).
 36. The method of claim 34, whereinthe at least one polynucleotide modification template generates amodified endogenous EPSPS gene encoding a plant EPSPS polypeptidecomprising A4W, H54M L98C, G102A, K173R I208L, K243E E302S, T361S,E391P, D402G, A416G, V438R, S440R, T441Q, and F442V.
 37. The method ofclaim 34, wherein the at least one polynucleotide modification templategenerates a modified endogenous EPSPS gene encoding a plant EPSPSpolypeptide comprising A2R, A4W, A72Q, K84R, L98C, G102A, I208L, T279A,E302S, T361S, E391G, D402G, A416G, V438R, and T441Q.
 38. The method ofclaim 34, wherein the at least one polynucleotide modification templategenerates a modified endogenous EPSPS gene encoding a plant EPSPSpolypeptide comprising A2R, A4W, K84R, L98C, K208L, K243E, E391P, andD402G.
 39. The method of claim 34, wherein the at least onepolynucleotide modification template generates a modified endogenousEPSPS gene encoding a plant EPSPS polypeptide having the amino acidsequence set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. 40.(canceled)
 41. (canceled)
 42. A glyphosate tolerant rice plantexpressing a plant EPSPS polypeptide comprising an amino acid mutationthat is analogous to G102A and at least one amino acid mutation that isanalogous to the amino acid mutation selected from the group consistingof: a) A2R, b) A4W, c) H54M, d) A72Q, e) K84R, f) L98C, g) K173R, h)I208L, i) K243E, j) T279A, k) E302S, l) T361S, m) E391P, n) E391G, o)D402G, p) A416G, q) V438R, r) S440R, s) T441Q, and t) F442V, whereineach amino acid mutation position corresponds to the analogous aminoacid position set forth in SEQ ID NO:1 and wherein the plant EPSPSpolypeptide comprises a sequence that is at least 90% identical to SEQID NO:7. 43-64. (canceled)
 65. The glyphosate tolerant rice plant ofclaim 42, wherein the plant EPSPS polypeptide comprises a sequence thatis at least 95% identical to SEQ ID NO:7.
 66. The glyphosate tolerantrice plant of claim 42, wherein a heterologous promoter is operablylinked to a polynucleotide encoding the EPSPS polypeptide.